Single-headed myosin II acts as a dominant negative mutation in Dictyostelium.

Burns, C. Geoffrey; Larochelle, Denis A.; Erickson, Harold P.; Reedy, Mary C.; Lozanne, Arturo De

doi:10.1073/pnas.92.18.8244

Cited by 21 publications

(23 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3). Based on equivalent modifications in Dictyostelium, mYFP- myosin II DN homodimers should completely lack actin binding and contractility, and the 'single headed' wild-type myosin/mYFP-myosin II DN heterodimers should have severely decreased actin binding and contractility (Burns et al, 1995;Uyeda and Yumura, 2000;Zang and Spudich, 1998). Consistent with this, we find that YFP-containing myosin isolated from mYFP-myosin II DN expressing Drosophila embryos shows reduced actin binding when compared with wild-type myosin in a standard spin down assay (see Fig.…”

Section: Functionally Distinct Modes Of Myosin Localizationsupporting

confidence: 70%

“…The PCR KpnI-XhoI central myosin fragment was inserted into the KpnI/XhoI-digested pBS-myosin-II-C-terminus backbone, to create myosin-II DN . This construct contains a dominant-negative form of myosin II, with the head removed at precisely the same point as an equivalent Dictyostelium myosin II DN [head-neck junction: amino acid 831 in Drosophila (QWWR) and position 809 in Dictyostelium (PWWK)] (Burns et al, 1995;Zang and Spudich, 1998). mYFP (Haseloff, 1999) (gift from J. Haseloff) was PCR amplified to introduce KpnI and NotI sites.…”

Section: Puast-myfp-myosin-ii Dnmentioning

confidence: 99%

See 1 more Smart Citation

folded gastrulation, cell shape change and the control of myosin localization

et al. 2005

View full text Add to dashboard Cite

The global cell movements that shape an embryo are driven by intricate changes to the cytoarchitecture of individual cells. In a developing embryo,these changes are controlled by patterning genes that confer cell identity. However, little is known about how patterning genes influence cytoarchitecture to drive changes in cell shape. In this paper, we analyze the function of the folded gastrulation gene (fog), a known target of the patterning gene twist. Our analysis of fog function therefore illuminates a molecular pathway spanning all the way from patterning gene to physical change in cell shape. We show that secretion of Fog protein is apically polarized, making this the earliest polarized component of a pathway that ultimately drives myosin to the apical side of the cell. We demonstrate that fog is both necessary and sufficient to drive apical myosin localization through a mechanism involving activation of myosin contractility with actin. We determine that this contractility driven form of localization involves RhoGEF2 and the downstream effector Rho kinase. This distinguishes apical myosin localization from basal myosin localization, which we find not to require actinomyosin contractility or FOG/RhoGEF2/Rho-kinase signaling. Furthermore, we demonstrate that once localized apically, myosin continues to contract. The force generated by continued myosin contraction is translated into a flattening and constriction of the cell surface through a tethering of the actinomyosin cytoskeleton to the apical adherens junctions. Our analysis of fog function therefore provides a direct link from patterning to cell shape change.

show abstract

Section: Functionally Distinct Modes Of Myosin Localizationsupporting

confidence: 70%

Section: Puast-myfp-myosin-ii Dnmentioning

confidence: 99%

folded gastrulation, cell shape change and the control of myosin localization

et al. 2005

View full text Add to dashboard Cite

show abstract

“…A n alternate possibility considered at the outset of this study was that myosin II would still be able to form bipolar thick filaments in these cells, but the incorporation of L M M molecules into these filaments would disrupt their normal function. This idea was stimulated by our finding that the formation of single-headed myosin II in Dictyostelium cells leads to its incorporation into existent myosin filaments in a manner that disrupts myosin function in vivo (Burns et al, 1995). In contrast, the results described here illustrate that overexpression of LMM totally disrupts normal myosin filament formation by sequestering the native myosin II into abnormal aggregates.…”

Section: Discussioncontrasting

confidence: 39%

“…We attempted to obtain cell lines that expressed LMM at a lower level, however, analysis of many independent transformants indicated that all expressed the same high levels of LMM. In contrast, the levels of expression of other myosin tail fragments ($2 and Rod fragments) using the same vector reached only a 1:1 molar ratio with respect to the endogenous MHC (Burns et al, 1995).…”

Section: Expression Of Lmmmentioning

confidence: 98%

Expression of light meromyosin in Dictyostelium blocks normal myosin II function.

Burns

Reedy

Heuser

et al. 1995

The Journal of Cell Biology

Self Cite

View full text Add to dashboard Cite

Abstract. The ability of myosin II to form filaments is essential for its function in vivo. This property of self association is localized in the light meromyosin (LMM) region of the myosin II molecules. To explore this property in more detail within the context of living cells, we expressed the LMM portion of the Dictyostelium myosin II heavy chain gene in wild-type Dictyostelium cells.We found that the LMM protein was expressed at high levels and that it folded properly into a-helical coiledcoiled molecules. The expressed LMM formed large cytoplasmic inclusions composed of entangled short filaments surrounded by networks of long tubular structures. Importantly, these abnormal structures sequestered the cell's native myosin II, completely removing it from its normal cytoplasmic distribution. As a result the cells expressing LMM displayed a myosinnull phenotype: they failed to undergo cytokinesis and became multinucleate, failed to form caps after treatment with Con A, and failed to complete their normal developmental cycle. Thus, expression of the LMM fragment in Dictyostelium completely abrogates myosin II function in vivo. The dominant-negative character of this phenotype holds promise as a general method to disrupt myosin II function in many cell types without the necessity of gene targeting. THE distribution of myosin II in nonmuscle cells is constantly changing according to the needs of the cells. During cytokinesis, for example, most myosin II assembles into filaments that localize to the contractile ring during anaphase and disassemble shortly thereafter upon completion of cell division. In a moving cell, myosin II resides in the trailing edge; however myosin must change its localization rapidly when a cell changes direction and forms a new trailing edge. One possible mechanism for such dramatic reorganization of myosin II molecules is via a precise regulation of myosin filament assembly in a spatial and temporal manner. A second possible means to change myosin II distribution would be to move the myosin II filaments along actin filaments to particular cellular locations. However, we currently know little about the relative contribution of filament assembly vs. filament movement to the spatial organization of myosin II in living cells.The tail portion of Dictyostelium myosin II contains two domains involved in filament formation and its regulation. The carboxyl-terminal 34-kD segment of the myosin tail (C-light meromyosin [LMM]-34) 1 contains three phosphorylation sites which regulate filament formation in re-

show abstract

“…In Dictyostelium, myosin function has been disrupted genetically either by replacing the single myosin heavy chain gene with a truncated form or by removing it entirely (De Lozanne and Spudich, 1987;Manstein et al, 1989). Alternatively, myosin function can be reduced significantly by expressing antisense RNA or by expressing the carboxy-terminal rod domain of the protein (Knecht and Loomis, 1987;Burns et al, 1995). Regard-less of the method of disrupting myosin function, the phenotype is similar: cells lacking myosin cannot undergo cytokinesis when grown in suspension culture.…”

Section: Introductionmentioning

confidence: 92%

Myosin Heavy Chain Phosphorylation Sites Regulate Myosin Localization during Cytokinesis in Live Cells

Sabry

Moores

Ryan

et al. 1997

MBoC

View full text Add to dashboard Cite

Conventional myosin II plays a fundamental role in the process of cytokinesis where, in the form of bipolar thick filaments, it is thought to be the molecular motor that generates the force necessary to divide the cell. In Dictyostelium, the formation of thick filaments is regulated by the phosphorylation of three threonine residues in the tail region of the myosin heavy chain. We report here on the effects of this regulation on the localization of myosin in live cells undergoing cytokinesis. We imaged fusion proteins of the green-fluorescent protein with wild-type myosin and with myosins where the three critical threonines had been changed to either alanine or aspartic acid. We provide evidence that thick filament formation is required for the accumulation of myosin in the cleavage furrow and that if thick filaments are overproduced, this accumulation is markedly enhanced. This suggests that myosin localization in dividing cells is regulated by myosin heavy chain phosphorylation.

show abstract

Single-headed myosin II acts as a dominant negative mutation in Dictyostelium.

Cited by 21 publications

References 23 publications

folded gastrulation, cell shape change and the control of myosin localization

folded gastrulation, cell shape change and the control of myosin localization

Expression of light meromyosin in Dictyostelium blocks normal myosin II function.

Myosin Heavy Chain Phosphorylation Sites Regulate Myosin Localization during Cytokinesis in Live Cells

Contact Info

Product

Resources

About