Dynamic assembly properties of nonmuscle myosin II isoforms revealed by combination of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

Mitsuhashi, Mariko; Sakata, Hiroshi; Kinjo, Masataka; Yazawa, Michio; Takahashi, Masayuki

doi:10.1093/jb/mvq134

Cited by 15 publications

(19 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Despite repeated efforts to answer the question of NMII heteropolymerization in vivo by biochemical or molecular biology approaches [19–21], the isoform composition of endogenous bipolar NMII filaments remained unknown. Our immunogold PREM data give an explicit answer to this question by showing individual double-labeled NMIIA/NMIIB bipolar filaments.…”

Section: Discussionmentioning

confidence: 99%

“…Such model implies that NMIIA and NMIIB more likely form homotypic filaments that sequentially incorporate into the contractile system. However, existing data suggest that muscle and nonmuscle myosins II can copolymerize in vitro [18], truncated NMIIA and NMIIB proteins can heteropolymerize in vitro [19] and in cells [20], and endogenous NMII isoforms can be coimmunoprecipitated from lysates [21]. However, explicit evidence for copolymerization of endogenous NMII isoforms into heterotypic bipolar filaments is not available and significance of such copolymerization, if it exists, is not known.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Endogenous Species of Mammalian Nonmuscle Myosin IIA and IIB Include Activated Monomers and Heteropolymers

et al. 2014

View full text Add to dashboard Cite

Summary Background Class II myosins generate contractile forces in cells by polymerizing into bipolar filaments and pulling on anchored actin filaments. Nonmuscle myosin II (NMII) plays central roles during cell adhesion, migration, cytokinesis, and tissue morphogenesis. NMII is present in virtually all mammalian cell types as tissue-specific combinations of NMIIA, NMIIB, and NMIIC isoforms. It remains poorly understood how the highly dynamic NMII-actin contractile system begins to assemble at new cellular locations during cell migration and how incorporation of different NMII isoforms into this system is coordinated. Results Using platinum replica electron microscopy in combination with immunogold labeling, we demonstrate that individual activated (phosphorylated on the regulatory light chain and unfolded) NMIIA and NMIIB molecules represent a functional form of NMII in motile cells and that NMIIA and NMIIB copolymerize into nascent bipolar filaments during contractile system assembly. Using subdiffraction STED microscopy together with a pharmacological block-and-release approach, we report that NMIIA and NMIIB simultaneously incorporate into the cytoskeleton during initiation of contractile system assembly, whereas the characteristic rearward shift of NMIIB relative to NMIIA is established later in the course of NMII turnover. Conclusions We show existence of activated NMII monomers in cells, copolymerization of endogenous NMIIA and NMIIB molecules, and contribution of both isoforms, rather than only NMIIA, to early stages of the contractile system assembly. These data change the current paradigms about dynamics and functions of NMII and provide new conceptual insights into organization and dynamics of the ubiquitous cellular machinery for contraction that acts in multiple cellular contexts.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Endogenous Species of Mammalian Nonmuscle Myosin IIA and IIB Include Activated Monomers and Heteropolymers

et al. 2014

View full text Add to dashboard Cite

show abstract

“…The metastasis factor mts1 also called S100A4 or calvasculin, a member of the S100 family of calcium-binding proteins, binds C-terminal ends of the MHC of myosin II. Binding of S100A4 to C-terminal ends of the MHC promotes phosphorylation on S1943 and disassembly of myosin II filamentation; however, the underlying mechanisms remain unknown to date (Li et al, 2003; Badyal et al, 2011; Mitsuhashi et al, 2011; Kiss et al, 2012). S100-P, another member of S100 family of calcium-binding proteins and a novel therapeutic target for cancer, interacts with myosin II in cells.…”

Section: Mhc Phosphorylation In Regulating Myosin II Activitymentioning

confidence: 99%

Life without double-headed non-muscle myosin II motor proteins

Betapudi

2014

Front. Chem.

View full text Add to dashboard Cite

Non-muscle myosin II motor proteins (myosin IIA, myosin IIB, and myosin IIC) belong to a class of molecular motor proteins that are known to transduce cellular free-energy into biological work more efficiently than man-made combustion engines. Nature has given a single myosin II motor protein for lower eukaryotes and multiple for mammals but none for plants in order to provide impetus for their life. These specialized nanomachines drive cellular activities necessary for embryogenesis, organogenesis, and immunity. However, these multifunctional myosin II motor proteins are believed to go awry due to unknown reasons and contribute for the onset and progression of many autosomal-dominant disorders, cataract, deafness, infertility, cancer, kidney, neuronal, and inflammatory diseases. Many pathogens like HIV, Dengue, hepatitis C, and Lymphoma viruses as well as Salmonella and Mycobacteria are now known to take hostage of these dedicated myosin II motor proteins for their efficient pathogenesis. Even after four decades since their discovery, we still have a limited knowledge of how these motor proteins drive cell migration and cytokinesis. We need to enrich our current knowledge on these fundamental cellular processes and develop novel therapeutic strategies to fix mutated myosin II motor proteins in pathological conditions. This is the time to think how to relieve the hijacked myosins from pathogens in order to provide a renewed impetus for patients' life. Understanding how to steer these molecular motors in proliferating and differentiating stem cells will improve stem cell based-therapeutics development. Given the plethora of cellular activities non-muscle myosin motor proteins are involved in, their importance is apparent for human life.

show abstract

“…DNA fragments encoding Leu 1666 -Glu 1961 of NMHC IIA (ARF296) and Phe 1672-Glu 1976 of NMHC IIB (BRF305) were amplified by PCR as described elsewhere. 31,32 Each of them was subcloned into the HindIII-BamH1 sites of pEGFP-C3 (Clontech) to generate the pEGFP-ARF296 and pEGFP-BRF305, respectively.…”

Section: Construction Of Plasmid Dnamentioning

confidence: 99%

“…Figure 7A illustrates the rod fragments, ARF296 and BRF305, which are encoded by Leu 1666-Glu 1961 of NMHC IIA and Phe 1672-Glu 1976 of NMHC IIB, respectively. 31,32 When ARF296 or BRF305 was exogenously expressed to CFU-E, these rods localized in cytoplasm ( Figure 7B) and blocked the proliferation of CFU-E ( Figure 7C). To investigate the function of myosin IIA and IIB in the enucleation of erythroblasts, mature erythroblasts (day 10 cells) were transfected with ARF296 and BRF305.…”

Section: Human Erythroblasts Express Non-muscle Myosin Iia and Iibmentioning

confidence: 99%

Enucleation of human erythroblasts involves non-muscle myosin IIB

Ubukawa

Guo

Takahashi

et al. 2012

Blood

Self Cite

View full text Add to dashboard Cite

Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various celldivision inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34 ؉ cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts. IntroductionDuring erythropoiesis, stem cells undergo lineage specific commitment and generate erythroid progenitor cells through cellular division events including nuclear (mitosis) and cytoplasmic (cytokinesis) division. These progenitor cells consist of immature and mature erythroid progenitors, the burst-forming unit-erythroid (BFU-E) and the colony-forming unit-erythroid (CFU-E), respectively. The BFU-E can be considered as a progenitor of the CFU-E. Indeed, after 6 to 7 days in culture, cells generated from human BFU-E have all the functional characteristics of CFU-E 1 . After an additional 6 to 7 days in culture, human CFU-E proliferate and differentiate into mature erythroblasts. 1 Terminally differentiated erythroblasts in mammals expel their nuclei via a process termed enucleation, becoming reticulocytes and subsequently mature erythrocytes. The nucleus separates from the remainder of the cell and is phagocytosed by reticular cells such as macrophages (for a review, see Chasis et al 2 ).Enucleation of erythroblasts is thought to occur through a process similar to cytokinesis. Several general principles apply to cytokinesis. Firstly, the microtubule cytoskeleton plays an important role in both the choice and positioning of the division site. Once this site is chosen, the local assembly of the actomyosin contractile ring remodels the plasma membrane. Finally, membrane trafficking to, and membrane fusion at the division site result in the physical separation of the daughter cells, a process termed abscission (for reviews, see Barr et al 3 and Glotzer et al 4 ). Although modulation of the actomyosin cytoskeleton is crucial for proper cytokinesis, there is a paucity of information regarding how non-muscle myosin II contributes to enucleation.Several investigations have studied the mol...

show abstract

Dynamic assembly properties of nonmuscle myosin II isoforms revealed by combination of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

Cited by 15 publications

References 53 publications

Endogenous Species of Mammalian Nonmuscle Myosin IIA and IIB Include Activated Monomers and Heteropolymers

Endogenous Species of Mammalian Nonmuscle Myosin IIA and IIB Include Activated Monomers and Heteropolymers

Life without double-headed non-muscle myosin II motor proteins

Enucleation of human erythroblasts involves non-muscle myosin IIB

Contact Info

Product

Resources

About