Georgios Tsiavaliaris

Molecular engineering of a backwards-moving myosin motor

All members of the diverse myosin superfamily have a highly conserved globular motor domain that contains the actin- and nucleotide-binding sites and produces force and movement. The light-chain-binding domain connects the motor domain to a variety of functionally specialized tail domains and amplifies small structural changes in the motor domain through rotation of a lever arm. Myosins move on polarized actin filaments either forwards to the barbed (+ ) or backwards to the pointed (- ) end. Here, we describe the engineering of an artificial backwards-moving myosin from three pre-existing molecular building blocks. These blocks are: a forward-moving class I myosin motor domain, a directional inverter formed by a four-helix bundle segment of human guanylate-binding protein-1 and an artificial lever arm formed by two α-actinin repeats. Our results prove that reverse-direction movement of myosins can be achieved simply by rotating the direction of the lever arm 180°.

The mechanism of pentabromopseudilin inhibition of myosin motor activity

We have identified pentabromopseudilin (PBP) as a potent inhibitor of myosin-dependent processes such as isometric tension development and unloaded shortening velocity. PBP-induced reductions in the rate constants for ATP binding, ATP hydrolysis and ADP dissociation extend the time required per myosin ATPase cycle in the absence and presence of actin. Additionally, coupling between the actin and nucleotide binding sites is reduced in the presence of the inhibitor. The selectivity of PBP differs from that observed with other myosin inhibitors. To elucidate the binding mode of PBP, we crystallized the Dictyostelium myosin-2 motor domain in the presence of Mg2+-ADP–meta-vanadate and PBP. The electron density for PBP is unambiguous and shows PBP to bind at a previously unknown allosteric site near the tip of the 50-kDa domain, at a distance of 16 Å from the nucleotide binding site and 7.5 Å away from the blebbistatin binding pocket.

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 Mg2+ 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 and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity

Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC50 values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway.

Expression vectors for studying cytoskeletal proteins in Dictyostelium discoideum

We generated and tested a set of cloning vectors designed to facilitate the production, purification and visualization of proteins in Dictyostelium discoideum. The vectors are derived from the Dictyostelium-E. coli shuttle vector pDXA-3H (6.1 kb), which carries the origin of replication of the Dd high-copy-number plasmid, Ddp2, a high-copy-number E. coli plasmid origin of replication, an act6 promoter driven G418 resistance cassette, the bacterial ampicillin resistance gene and an expression cassette. The new cloning vectors carry expression cassettes consisting of the strong constitutive actin-15 promoter, a translation start followed by a multiple cloning site, sequences for the addition of purification or visualization tags, and Dictyostelium polyadenylation and termination signals. Vectors designed to facilitate protein visualization in living Dictyostelium cells contain either coding sequences for the cyan (CFP) or yellow (YFP) variants of green fluorescent protein (GFP). Versions of the vectors for the production of N- and C-terminal fusions with the fluorescent proteins were generated. To facilitate protein purification, vectors for the production of glutathione-S-transferase (GST) fusion proteins and Strep- or FLAG-affinity-tagged proteins were generated. Additionally, a vector for the production of His8-tagged proteins was generated, which has the G418-resistance cassette replaced by a hygromycin resistance cassette.

A Myosin IK-Abp1-PakB Circuit Acts as a Switch to Regulate Phagocytosis Efficiency

Actin dynamics and myosin contractile forces are necessary to form and close the phagocytic cup. A myosin I, MyoK, a myosin-Arp2/3 linker, Abp1, and a Rac-dependent kinase, PakB form a circuit that regulates phagocytosis. MyoK is phosphorylated by PakB and positively regulates uptake, whereas binding of Abp1 negatively regulates PakB and MyoK.

Total synthesis of biologically active alkaloids using transition metals

We have developed efficient synthetic routes to heterocyclic ring systems using transition metals (palladium, iron, and silver). Recent applications of this chemistry to the total synthesis of biologically active alkaloids include carbazole alkaloids (pityriazole, euchrestifoline, the antiostatins), crispine A, pentabromo- and pentachloropseudilin. The two latter alkaloids represent a novel class of myosin ATPase inhibitors that led to the discovery of a new allosteric binding site of the protein.

Dictyostelium Myosin-5b Is a Conditional Processive Motor

Dictyostelium myosin-5b is the gene product of myoJ and one of two closely related myosin-5 isoenzymes produced in Dictyostelium discoideum. Here we report a detailed investigation of the kinetic and functional properties of the protein. In standard assay buffer conditions, Dictyostelium myosin-5b displays high actin affinity in the presence of ADP, fast ATP hydrolysis, and a high steady-state ATPase activity in the presence of actin that is rate limited by ADP release. These properties are typical for a processive motor that can move over long distances along actin filaments without dissociating. Our results show that a physiological decrease in the concentration of free Mg2+-ions leads to an increased rate of ADP release and shortening of the fraction of time the motor spends in the strong actin binding states. Consistently, the ability of the motor to efficiently translocate actin filaments at very low surface densities decreases with decreasing concentrations of free Mg2+-ions. In addition, we provide evidence that the observed changes in Dd myosin-5b motor activity are of physiological relevance and propose a mechanism by which this molecular motor can switch between processive and non-processive movement.

Functional characterization of the N-terminal region of myosin-2.

All class 2 myosins contain an N-terminal extension of ∼80 residues that includes an Src homology 3 (SH3)-like subdomain. To explore the functional importance of this region, which is also present in most other myosin classes, we generated truncated constructs of Dictyostelium discoideum myosin-2. Truncation at position 80 resulted in the complete loss of myosin-2 function in vivo. Actin affinity was more than 80-fold, and the rate of ADP release ∼40-fold decreased in this mutant. In contrast, a myosin construct that lacks only the SH3-like subdomain, corresponding to residues 33-79, displayed much smaller functional defects. In complementation experiments with myosin-2 null cells, this construct rescued myosin-2-dependent processes such as cytokinesis, fruiting body formation, and sporogenesis. An 8-fold reduction in motile activity and changes of similar extent in the affinity for ADP and filamentous actin indicate the importance of the SH3-like subdomain for correct communication between the functional regions within the myosin motor domain and suggest that local perturbations in this region can play a role in modulating myosin-2 motor activity.

Myosin-1C associates with microtubules and stabilizes the mitotic spindle during cell division

The mitotic spindle in eukaryotic cells is composed of a bipolar array of microtubules (MTs) and associated proteins that are required during mitosis for the correct partitioning of the two sets of chromosomes to the daughter cells. In addition to the well-established functions of MT-associated proteins (MAPs) and MT-based motors in cell division, there is increasing evidence that the F-actin-based myosin motors are important mediators of F-actin–MT interactions during mitosis. Here, we report the functional characterization of the long-tailed class-1 myosin myosin-1C from Dictyostelium discoideum during mitosis. Our data reveal that myosin-1C binds to MTs and has a role in maintenance of spindle stability for accurate chromosome separation. Both myosin-1C motor function and tail-domain-mediated MT–F-actin interactions are required for the cell-cycle-dependent relocalization of the protein from the cell periphery to the spindle. We show that the association of myosin-1C with MTs is mediated through the tail domain. The myosin-1C tail can inhibit kinesin motor activity, increase the stability of MTs, and form crosslinks between MTs and F-actin. These data illustrate that myosin-1C is involved in the regulation of MT function during mitosis in D. discoideum.