Thomas T. Egelhoff

Dictyostelium myosin heavy chain phosphorylation sites regulate myosin filament assembly and localization in vivo.

Three threonine residues in the tail region of Dictyostelium myosin II heavy chain have been implicated previously in control of myosin filament formation. Here we report the in vitro and in vivo consequences of converting these sites to alanine residues, which eliminates phosphorylation at these positions, or to aspartate residues, which mimics the negative charge state of the phosphorylated molecule. Alanine substitution allows in vitro assembly and in vivo contractile activity, although this myosin shows substantial over-assembly in vivo. Aspartate substitution eliminates filament assembly in vitro and renders the myosin unable to drive any tested contractile event in vivo. These results demonstrate that heavy chain phosphorylation plays a key modulatory role in controlling myosin function in vivo.

Distinct Roles of Nonmuscle Myosin II Isoforms in the Regulation of MDA-MB-231 Breast Cancer Cell Spreading and Migration

Initial stages of tumor cell metastasis involve an epithelial-mesenchyme transition that involves activation of amoeboid migration and loss of cell-cell adhesion. The actomyosin cytoskeleton has fundamental but poorly understood roles in these events. Myosin II, an abundant force-producing protein, has roles in cell body translocation and retraction of the posterior of the cell during migration. Recent studies have suggested that this protein may also have roles in leading edge protrusive events. The metastasis-promoting protein metastasin-1, a regulator of myosin II assembly, colocalizes with myosin IIA at the leading edge of cancer cells, suggesting direct roles for myosin II in metastatic behavior. We have assessed the roles of specific myosin II isoforms during lamellar spreading of MDA-MB-231 breast cancer cells on extracellular matrix. We find that the two major myosin II isoforms IIA and IIB are both expressed in these cells, and both are recruited dramatically to the lamellar margin during active spreading on fibronectin. There is also a transient increase in regulatory light chain phosphorylation that correlates the recruitment of myosin IIA and myosin IIB into this spreading margin. Pharmacologic inhibition of myosin II or myosin light chain kinase dramatically reduced spreading. Depletion of myosin IIA via small interfering RNA impaired migration but enhanced lamellar spreading, whereas depletion of myosin IIB impaired not only migration but also impaired initial rates of lamellar spreading. These results indicate that both isoforms are critical for the mechanics of cell migration, with myosin IIB seeming to have a preferential role in the mechanics of lamellar protrusion.

Recruitment of a myosin heavy chain kinase to actin-rich protrusions in Dictyostelium.

Nonmuscle myosin II plays fundamental roles in cell body translocation during migration and is typically depleted or absent from actin-based cell protrusions such as lamellipodia, but the mechanisms preventing myosin II assembly in such structures have not been identified [1-3]. In Dictyostelium discoideum, myosin II filament assembly is controlled primarily through myosin heavy chain (MHC) phosphorylation. The phosphorylation of sites in the myosin tail domain by myosin heavy chain kinase A (MHCK A) drives the disassembly of myosin II filaments in vitro and in vivo [4]. To better understand the cellular regulation of MHCK A activity, and thus the regulation of myosin II filament assembly, we studied the in vivo localization of native and green fluorescent protein (GFP)-tagged MHCK A. MHCK A redistributes from the cytosol to the cell cortex in response to stimulation of Dictyostelium cells with chemoattractant in an F-actin-dependent manner. During chemotaxis, random migration, and phagocytic/endocytic events, MHCK A is recruited preferentially to actin-rich leading-edge extensions. Given the ability of MHCK A to disassemble myosin II filaments, this localization may represent a fundamental mechanism for disassembling myosin II filaments and preventing localized filament assembly at sites of actin-based protrusion.

Phosphorylation of the Dictyostelium myosin II heavy chain is necessary for maintaining cellular polarity and suppressing turning during chemotaxis

Conversion of the three mapped threonine phosphorylation sites in the myosin II heavy chain tail to alanines results in a mutant (3XALA) in Dictyostelium discoideum, which displays constitutive myosin overassembly in the cytoskeleton and increased cortical tension. To assess the importance of myosin phosphorylation in cellular translocation and chemotaxis, 3XALA mutant cells have been analyzed by 2D and 3D computer-assisted methods in buffer, in a spatial gradient of cAMP, and after the rapid addition of cAMP. 3XALA cells crawling in buffer exhibit distinct abnormalities in cellular shape, the maintenance of polarity and the complexity of the pseudopod perimeter. 3XALA cells crawling in buffer also exhibit a decrease in directionality. In a spatial gradient of cAMP, the behavioral defects are accentuated. In a spatial gradient, 3XALA cells exhibit a repeating 1- to 2-min behavior cycle in which the shape of each cell changes abnormally from elongate to extremely wide with lateral, opposing pseudopods. At the end of each cycle, 3XALA cells turn 90 degrees into the left or right lateral pseudopod, resulting in a dramatic depression in chemotactic efficiency, even though 3XALA cells are chemotactically responsive to cAMP. These results demonstrate that the phosphorylation of myosin II heavy chain plays a critical role in the maintenance of cell shape and in persistent translocation in a spatial gradient of chemoattractant.

Molecular genetic tools for study of the cytoskeleton in Dictyostelium

Publisher Summary Dictyostelium discoideum has a number of features that make it an attractive system for cell biological studies. The ability of Dictyostelium cells to perform active ameboid crawling and chemotaxis has made it a popular system for cell motility and signal transduction studies. The lack of a cell wall and ease of cultivation make Dictyostelium an excellent organism for biochemical approaches, allowing large quantities of material to be obtained and lysed without difficulty. This chapter describes molecular genetic tools that are used for transformation, construction of null cell lines, and expression of the cloned myosin gene fragments. Brief coverage is given to other tools and methods common in the field, but the emphasis is on those approaches that are currently being used. Straightforward gene disruption protocols have been developed and used to construct myosin null lines of Dictyostelium . These techniques have also been successfully applied to the study of other genes in Dictyostelium , such as the α-actinin gene.

Multiple Regulatory Steps Control Mammalian Nonmuscle Myosin II Assembly in Live Cells

To better understand the mechanism controlling nonmuscle myosin II (NM-II) assembly in mammalian cells, mutant NM-IIA constructs were created to allow tests in live cells of two widely studied models for filament assembly control. A GFP-NM-IIA construct lacking the RLC binding domain (DeltaIQ2) destabilizes the 10S sequestered monomer state and results in a severe defect in recycling monomers during spreading, and from the posterior to the leading edge during polarized migration. A GFP-NM-IIA construct lacking the nonhelical tailpiece (Deltatailpiece) is competent for leading edge assembly, but overassembles, suggesting defects in disassembly from lamellae subsequent to initial recruitment. The Deltatailpiece phenotype was recapitulated by a GFP-NM-IIA construct carrying a mutation in a mapped tailpiece phosphorylation site (S1943A), validating the importance of the tailpiece and tailpiece phosphorylation in normal lamellar myosin II assembly control. These results demonstrate that both the 6S/10S conformational change and the tailpiece contribute to the localization and assembly of myosin II in mammalian cells. This work furthermore offers cellular insights that help explain platelet and leukocyte defects associated with R1933-stop alleles of patients afflicted with human MYH9-related disorder.

Mapping of the Novel Protein Kinase Catalytic Domain of Dictyostelium Myosin II Heavy Chain Kinase A

Myosin heavy chain kinase A (MHCK A) in Dictyostelium was identified as a biochemical activity that phosphorylates threonine residues in the myosin II tail domain and regulates myosin filament assembly. The catalytic domain of MHCK A has now been mapped through the functional characterization of a series of MHCK A truncation mutants expressed in Escherichia coli A recombinant protein comprising the central nonrepetitive domain of MHCK A (residues 552-841) was isolated in a soluble form and shown to phosphorylate Dictyostelium myosin II, myelin basic protein, and a synthetic peptide substrate. The functionally mapped catalytic domain of MHCK A shows no detectable sequence similarity to known classes of eukaryotic protein kinases but shares substantial sequence similarity with a transcribed Caenorhabditis elegans gene and with the mammalian elongation factor-2 kinase (calcium/calmodulin-dependent protein kinase III). We suggest that MHCK A represents the prototype for a novel, widely occurring protein kinase family.

Signaling pathways regulating Dictyostelium myosin II

Dictyostelium myosin II is a conventional, two-headed myosin that consists of two copies each of a myosin heavy chain (MHC), an essential light chain (ELC) and a regulatory light chain (RLC). The MHC is comprised of an amino-terminal motor domain, a neck region that binds the RLC and ELC and a carboxyl-terminal α-helical coiled-coil tail. Electrostatic interactions between the tail domains mediate the self-assembly of myosin II into bipolar filaments that are capable of interacting with actin filaments to generate a contractile force. In this review we discuss the regulation of Dictyostelium myosin II by a myosin light chain kinase (MLCK-A) that phosphorylates the RLC and increases motor activity and by MHC kinases (MHCKs) that phosphorylate the tail and prevent filament assembly. Dictyostelium may express as many as four MHCKs (MHCK A–D) consisting of an atypical α-kinase catalytic domain and a carboxyl-terminal WD repeat domain that targets myosin II filaments. A previously reported MHCK, termed MHC-PKC, now seems more likely to be a diacylglycerol kinase (DgkA). The relationship of the MHCKs to the larger family of α-kinases is discussed and key features of the structure of the α-kinase catalytic domain are reviewed. Potential upstream regulators of myosin II are described, including DgkA, cGMP, cAMP and PAKa, a target for Rac GTPases. Recent results point to a complex network of signaling pathways responsible for controling the activity and localization of myosin II in the cell.

Myosin II isoform switching mediates invasiveness after TGF-β–induced epithelial–mesenchymal transition

Despite functional significance of nonmuscle myosin II in cell migration and invasion, its role in epithelial–mesenchymal transition (EMT) or TGF-β signaling is unknown. Analysis of normal mammary gland expression revealed that myosin IIC is expressed in luminal cells, whereas myosin IIB expression is up-regulated in myoepithelial cells that have more mesenchymal characteristics. Furthermore, TGF-β induction of EMT in nontransformed murine mammary gland epithelial cells results in an isoform switch from myosin IIC to myosin IIB and increased phosphorylation of myosin heavy chain (MHC) IIA on target sites known to regulate filament dynamics (S1916, S1943). These expression and phosphorylation changes are downstream of heterogeneous nuclear ribonucleoprotein-E1 (E1), an effector of TGF-β signaling. E1 knockdown drives cells into a migratory, invasive mesenchymal state and concomitantly up-regulates MHC IIB expression and MHC IIA phosphorylation. Abrogation of myosin IIB expression in the E1 knockdown cells has no effect on 2D migration but significantly reduced transmigration and macrophage-stimulated collagen invasion. These studies indicate that transition between myosin IIC/myosin IIB expression is a critical feature of EMT that contributes to increases in invasive behavior.

Differential localization in cells of myosin II heavy chain kinases during cytokinesis and polarized migration

BackgroundCortical myosin-II filaments in Dictyostelium discoideum display enrichment in the posterior of the cell during cell migration and in the cleavage furrow during cytokinesis. Filament assembly in turn is regulated by phosphorylation in the tail region of the myosin heavy chain (MHC). Early studies have revealed one enzyme, MHCK-A, which participates in filament assembly control, and two other structurally related enzymes, MHCK-B and -C. In this report we evaluate the biochemical properties of MHCK-C, and using fluorescence microscopy in living cells we examine the localization of GFP-labeled MHCK-A, -B, and -C in relation to GFP-myosin-II localization.ResultsBiochemical analysis indicates that MHCK-C can phosphorylate MHC with concomitant disassembly of myosin II filaments. In living cells, GFP-MHCK-A displayed frequent enrichment in the anterior of polarized migrating cells, and in the polar region but not the furrow during cytokinesis. GFP-MHCK-B generally displayed a homogeneous distribution. In migrating cells GFP-MHCK-C displayed posterior enrichment similar to that of myosin II, but did not localize with myosin II to the furrow during the early stage of cytokinesis. At the late stage of cytokinesis, GFP-MHCK-C became strongly enriched in the cleavage furrow, remaining there through completion of division.ConclusionMHCK-A, -B, and -C display distinct cellular localization patterns suggesting different cellular functions and regulation for each MHCK isoform. The strong localization of MHCK-C to the cleavage furrow in the late stages of cell division may reflect a mechanism by which the cell regulates the progressive removal of myosin II as furrowing progresses.