F-Actin Bending Facilitates Net Actomyosin Contraction By Inhibiting Expansion With Plus-End-Located Myosin Motors

We investigate whether a microscopic system of two semi-flexible actin filaments with an attached myosin motor can facilitate contraction. Based on energy minimisation, we derive and analyse a partial differential equation model for a two-filament-motor structure embedded within a dense, two-dimensional network. Our method enables calculation of the plane stress tensor, providing a measure for contractility. After deriving the model, we use a combination of asymptotic analysis and numerical solutions to show how F-actin bending facilitates net contraction as a myosin motor traverses two symmetric filaments. Myosin motors close to the minus-ends facilitate contraction, whereas motors close to the plus-ends facilitate expansion. The leading-order solution for rigid filaments exhibits polarity-reversal symmetry, such that the contractile and expansive components balance to zero. Surprisingly, after introducing bending the first-order correction to stress indicates expansion. However, numerical solutions show that filament bending induces a geometric asymmetry that brings the filaments closer to parallel as a myosin motor approaches their plus-ends. This decreases the effective spring force opposing motion of the motor, enabling it to move faster close to filament plus-ends. This reduces the contribution of expansive stress, giving rise to net contraction. Further numerical solutions confirm that this applies beyond the small bending regime considered in the asymptotic analysis. Our findings confirm that filament bending gives rise to microscopic-scale actomyosin contraction, and provides a possible explanation for network-scale contraction.

Blocking Palmitoylation of Toxoplasma gondii Myosin Light Chain 1 Disrupts Glideosome Composition but Has Little Impact on Parasite Motility

ABSTRACT Toxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in immunocompromised individuals and the developing fetus. Like other apicomplexans, T. gondii uses an unusual form of substrate-dependent gliding motility to invade cells of its hosts and to disseminate throughout the body during infection. It is well established that a myosin motor consisting of a class XIVa heavy chain (TgMyoA) and two light chains (TgMLC1 and TgELC1/2) plays an important role in parasite motility. The ability of the motor to generate force at the parasite periphery is thought to be reliant upon its anchoring and immobilization within a peripheral membrane-bound compartment, the inner membrane complex (IMC). The motor does not insert into the IMC directly; rather, this interaction is believed to be mediated by the binding of TgMLC1 to the IMC-anchored protein, TgGAP45. Therefore, the binding of TgMLC1 to TgGAP45 is considered a key element in the force transduction machinery of the parasite. TgMLC1 is palmitoylated, and we show here that palmitoylation occurs on two N-terminal cysteine residues, C8 and C11. Mutations that block TgMLC1 palmitoylation completely abrogate the binding of TgMLC1 to TgGAP45. Surprisingly, the loss of TgMLC1 binding to TgGAP45 in these mutant parasites has little effect on their ability to initiate or sustain movement. These results question a key tenet of the current model of apicomplexan motility and suggest that our understanding of gliding motility in this important group of human and animal pathogens is not yet complete. IMPORTANCE Gliding motility plays a central role in the life cycle of T. gondii and other apicomplexan parasites. The myosin motor thought to power motility is essential for virulence but distinctly different from the myosins found in humans. Consequently, an understanding of the mechanism(s) underlying parasite motility and the role played by this unusual myosin may reveal points of vulnerability that can be targeted for disease prevention or treatment. We show here that mutations that uncouple the motor from what is thought to be a key structural component of the motility machinery have little impact on parasite motility. This finding runs counter to predictions of the current, widely held “linear motor” model of motility, highlighting the need for further studies to fully understand how apicomplexan parasites generate the forces necessary to move into, out of, and between cells of the hosts they infect.

Actin and an unconventional myosin motor, TgMyoF, control the organization and dynamics of the endomembrane network in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that relies on three distinct secretory organelles, the micronemes, rhoptries, and dense granules, for parasite survival and disease pathogenesis. Secretory proteins destined for these organelles are synthesized in the endoplasmic reticulum (ER) and sequentially trafficked through a highly polarized endomembrane network that consists of the Golgi and multiple post-Golgi compartments. Currently, little is known about how the parasite cytoskeleton controls the positioning of the organelles in this pathway, or how vesicular cargo is trafficked between organelles. Here we show that F-actin and an unconventional myosin motor, TgMyoF, control the dynamics and organization of the organelles in the secretory pathway, specifically ER tubule movement, apical positioning of the Golgi and post-Golgi compartments, apical positioning of the rhoptries, and finally, the directed transport of Rab6-positive and Rop1-positive vesicles. Thus, this study identifies TgMyoF and actin as the key cytoskeletal components that organize the endomembrane system in T. gondii.

Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity

Actin is a major intracellular protein with key functions in cellular motility, signaling and structural rearrangements. Its dynamic behavior, such as polymerisation and depolymerisation of actin filaments in response to intra- and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin-binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.

Actin and an unconventional myosin motor, TgMyoF control the organization and dynamics of the endomembrane network in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that relies on three distinct secretory organelles, the micronemes, rhoptries and dense granules, for parasite survival and disease pathogenesis. Secretory proteins destined for these organelles are synthesized in the endoplasmic reticulum (ER) and sequentially trafficked through a highly polarized endomembrane network that consists of the Golgi and multiple post-Golgi compartments. Currently, little is known about how the parasite cytoskeleton controls the positioning of the organelles in this pathway, or how vesicular cargo is trafficked between organelles. Here we show that F-actin and an unconventional myosin motor, TgMyoF, control the dynamics and organization of the organelles in the secretory pathway, specifically ER tubule movement, apical positioning of the Golgi and post-Golgi compartments, apical positioning of the rhoptries and finally, the directed transport of Rab6-positive and Rop1-positive vesicles. Thus, this study identifies TgMyoF and actin as the key cytoskeletal components that organize the endomembrane system in T. gondii.Author SummaryEndomembrane trafficking is a vital cellular process in all eukaryotic cells. In most cases the molecular motors myosin, kinesin and dynein transport cargo including vesicles, organelles and transcripts along actin and microtubule filaments in a manner analogous to a train moving on its tracks. For the unicellular eukaryote Toxoplasma gondii, the accurate trafficking of proteins through the endomembrane system is vital for parasite survival and pathogenicity. However, the mechanisms of cargo transport in this parasite are poorly understood. In this study, we fluorescently labeled multiple endomembrane organelles and imaged their movements using live cell microscopy. We demonstrate that filamentous actin and an unconventional myosin motor named TgMyoF control both the positioning of organelles in this pathway and the movement of transport vesicles throughout the parasite cytosol. This data provides new insight into the mechanisms of cargo transport in this important pathogen and expands are understanding of the biological roles of actin in the intracellular phase of the parasite’s growth cycle.

Conformational distributions of isolated myosin motor domains encode their mechanochemical properties

Myosin motor domains perform an extraordinary diversity of biological functions despite sharing a common mechanochemical cycle. Motors are adapted to their function, in part, by tuning the thermodynamics and kinetics of steps in this cycle. However, it remains unclear how sequence encodes these differences, since biochemically distinct motors often have nearly indistinguishable crystal structures. We hypothesized that sequences produce distinct biochemical phenotypes by modulating the relative probabilities of an ensemble of conformations primed for different functional roles. To test this hypothesis, we modeled the distribution of conformations for 12 myosin motor domains by building Markov state models (MSMs) from an unprecedented two milliseconds of all-atom, explicit-solvent molecular dynamics simulations. Comparing motors reveals shifts in the balance between nucleotide-favorable and nucleotide-unfavorable P-loop conformations that predict experimentally measured duty ratios and ADP release rates better than sequence or individual structures. This result demonstrates the power of an ensemble perspective for interrogating sequence-function relationships.