Dictyostelium mutants lacking multiple classic myosin I isoforms reveal combinations of shared and distinct functions (original) (raw)
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Dictyostelium myosin-IE is a fast molecular motor involved in phagocytosis
Class I myosins are single-headed motor proteins, implicated in various motile processes including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Here we describe the cellular localization of myosin-IE and its role in the phagocytic uptake of solid particles and cells. A complete analysis of the kinetic and motor properties of Dictyostelium discoideum myosin-IE was achieved by the use of motor domain constructs with artificial lever arms. Class I myosins belonging to subclass IC like myosin-IE are thought to be tuned for tension maintenance or stress sensing. In contrast to this prediction, our results show myosin-IE to be a fast motor. Myosin-IE motor activity is regulated by myosin heavy chain phosphorylation, which increases the coupling efficiency between the actin and nucleotide binding sites tenfold and the motile activity more than fivefold. Changes in the level of free Mg(2+) ions, which are within the physiological range, are shown to modulate the motor activity of myosin-IE by inhibiting the release of adenosine diphosphate.
Mechanism, Regulation, and Functional Properties of Dictyostelium Myosin-1B
Journal of Biological Chemistry, 2008
Myosin-1B is one of three long tailed class-1 myosins containing an ATP-insensitive actin-binding site in the tail region that are produced in Dictyostelium discoideum. Myosin-1B localizes to actin-rich structures at the leading edge of migrating cells where it has been implicated in the formation and retraction of membrane projections, the recycling of plasma membrane components, and intracellular particle transport. Here, we have used a combination of molecular engineering approaches to describe the kinetic and motile properties of the myosin-1B motor and its regulation by TEDS site phosphorylation. Our results show that myosin-1B is a low duty ratio motor and displays the fastest nucleotide binding kinetics of any of the Dictyostelium class-1 myosins studied so far. Different from Dictyostelium myosin-1D and myosin-1E, dephosphorylated myosin-1B is not inactivated but moves actin filaments efficiently, albeit at an up to 8-fold slower velocity in the in vitro motility assay. A further difference is that myosin-1B lacks the ability to switch between rapid movement and bearing tension upon physiological changes of free Mg 2؉ ions. In this respect, its motor properties appear to be more closely related to Dictyostelium myosin-2 and rabbit skeletal muscle myosin.
Signaling pathways regulating Dictyostelium myosin II
Journal of muscle research and cell motility, 2002
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 alpha-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 alpha-kinase catalytic domain and a carboxyl-terminal WD repeat domain that targets myosin II filaments. A previousl...
Embo Reports, 2002
based on the specific impairment of a gene product by the coexpression of a mutant version of the same gene product. We describe the detailed characterization of two myosin constructs containing either point mutations F487A or F506G in the relay region. Dictyostelium cells transformed with F487A or F506G myosin are unable to undergo processes that require myosin II function, including fruiting-body formation, normal cytokinesis and growth in suspension. Our results show that the dominantnegative inhibition of myosin function is caused by disruption of the communication between active site and lever arm, which blocks motor activity completely, and perturbation of the communication between active site and actin-binding site, leading to an ∼100-fold increase in the mutants' affinity for actin in the presence of ATP. + Present address:
Dictyostelium Myosin II Is Regulated during Chemotaxis by a Novel Protein Kinase C
Journal of Biological Chemistry, 1996
The myosin II heavy chain (MHC)-specific protein kinase C (MHC-PKC) isolated from Dictyostelium discoideum has been implicated in the regulation of myosin II assembly in response to the chemoattractant, cAMP (Ravid, S., and Spudich, J. A. (1989) J. Biol. Chem. 264, 15144-15150). Here we report that elimination of MHC-PKC results in the abolishment of MHC phosphorylation in response to cAMP. Cells devoid of MHC-PKC exhibit substantial myosin II overassembly, as well as aberrant cell polarization, chemotaxis, and morphological differentiation. Cells overexpressing the MHC-PKC contain highly phosphorylated MHC and exhibit impaired myosin II localization and no apparent cell polarization and chemotaxis. The results presented here provide direct evidence that MHC-PKC phosphorylates MHC in response to cAMP and plays an important role in the regulation of myosin II localization during chemotaxis.
Dictyostelium discoideum Myosin II: Characterization of Functional Myosin Motor Fragments
Biochemistry, 1997
The transient kinetic properties of the recombinant myosin head fragments M761 and M781, which both lack the light chain binding domain (LCBD) and correspond to the first 761 and 781 residues of Dictyostelium discoideum myosin II, were compared with those of the subfragment 1-like fragment M864 and a shorter catalytic domain fragment M754. The properties of M761, M781, and M864 are almost identical in regard to nucleotide binding, nucleotide hydrolysis, actin binding, and the interactions between actin and nucleotide binding sites. Only the rate of the hydrolysis step was significantly faster for M761 and the affinity of M781 for actin significantly weaker than for M864. This indicates that the LCBD plays no major role in the biochemical behavior of the myosin head. In contrast, loss of the peptide between 754 and 761 produced several major changes in the property of M754 as documented previously [Woodward, S. K. A., Geeves, M. A., & Manstein, D. J. (1995) Biochemistry 34, 16056-16064]. We further show that C-terminal extension of M761 with one or two R-actinin repeats has very little effect on the behavior of the protein. The recombinant nature of M761 and the fact that it can be produced and purified in large amounts make it an ideal construct for systematic studies of the structure, kinetics, and function of the myosin motor.
Journal of cell science, 1999
Cytoplasmic myosin II accumulates in the cleavage furrow and provides the force for cytokinesis in animal and amoeboid cells. One model proposes that a specific domain in the myosin II tail is responsible for its localization, possibly by interacting with a factor concentrated in the equatorial region. To test this possibility, we have expressed myosins carrying mutations in the tail domain in a strain of Dictyostelium cells from which the endogenous myosin heavy chain gene has been deleted. The mutations used in this study include four internal tail deletions: Mydelta824-941, Mydelta943-1464, Mydelta943-1194 and Mydelta1156-1464. Contrary to the prediction of the hypothesis, immunofluorescence staining demonstrated that all mutant myosins were able to move toward the furrow region. Chimeric myosins, which consisted of a Dictyostelium myosin head and chicken skeletal myosin tail, also efficiently localized to the cleavage furrow. All these deletion and chimeric mutant myosins, excep...