Secondary Structure and X-ray Crystallographic Analysis of the Glideosome-Associated Connector (GAC) from Toxoplasma gondii

A model for parasitic motility has been proposed in which parasite filamentous actin (F-actin) is attached to surface adhesins by a large component of the glideosome, known as the glideosome-associated connector protein (GAC). This large 286 kDa protein interacts at the cytoplasmic face of the plasma membrane with the phosphatidic acid-enriched inner leaflet and cytosolic tails of surface adhesins to connect them to the parasite actomyosin system. GAC is observed initially to the conoid at the apical pole and re-localised with the glideosome to the basal pole in gliding parasite. GAC presumably functions in force transmission to surface adhesins in the plasma membrane and not in force generation. Proper connection between F-actin and the adhesins is as important for motility and invasion as motor operation itself. This notion highlights the need for new structural information on GAC interactions, which has eluded the field since its discovery. We have obtained crystals that diffracted to 2.6–2.9 Å for full-length GAC from Toxoplasma gondii in native and selenomethionine-labelled forms. These crystals belong to space group P212121; cell dimensions are roughly a = 119 Å, b = 123 Å, c = 221 Å, α = 90°, β = 90° and γ = 90° with 1 molecule per asymmetric unit, suggesting a more compact conformation than previously proposed

MiR-320-3p Regulates the Proliferation and Differentiation of Myogenic Progenitor Cells by Modulating Actin Remodeling

Skeletal myogenesis is essential for the maintenance of muscle quality and quantity, and impaired myogenesis is intimately associated with muscle wasting diseases. Although microRNA (miRNA) plays a crucial role in myogenesis and relates to muscle wasting in obesity, the molecular targets and roles of miRNAs modulated by saturated fatty acids (SFA) are largely unknown. In the present study, we investigated the role of miR-320-3p on the differentiation of myogenic progenitor cells. Palmitic acid (PA), the most abundant dietary SFA, suppressed myogenic factors expression and impaired differentiation in C2C12 myoblasts, and these effects were accompanied by CFL2 downregulation and miR-320-3p upregulation. In particular, miR-320-3p appeared to target CFL2 mRNA directly and suppress the expression of CFL2, an essential factor for filamentous actin (F-actin) depolymerization. Transfection of myoblasts with miR-320-3p mimic increased F-actin formation and nuclear translocation of Yes-associated protein 1 (YAP1), a key component of mechanotransduction. Furthermore, miR-320-3p mimic increased myoblast proliferation and markedly impeded the expression of MyoD and MyoG, consequently inhibiting myoblast differentiation. In conclusion, our current study highlights the role of miR-320-3p on CFL2 expression, YAP1 activation, and myoblast differentiation and suggests that PA-inducible miR-320-3p is a significant mediator of muscle wasting in obesity.

Filamin A Is Required for NK Cell Cytotoxicity at the Expense of Cytokine Production via Synaptic Filamentous Actin Modulation

Natural killer (NK) cells are innate cytotoxic lymphocytes that efficiently eliminate malignant and virus-infected cells without prior activation via the directed and focused release of lytic granule contents for target cell lysis. This cytolytic process is tightly regulated at discrete checkpoint stages to ensure the selective killing of diseased target cells and is highly dependent on the coordinated regulation of cytoskeletal components. The actin-binding protein filamin crosslinks cortical actin filaments into orthogonal networks and links actin filament webs to cellular membranes to modulate cell migration, adhesion, and signaling. However, its role in the regulation of NK cell functions remains poorly understood. Here, we show that filamin A (FLNa), a filamin isoform with preferential expression in leukocytes, is recruited to the NK cell lytic synapse and is required for NK cell cytotoxicity through the modulation of conjugate formation with target cells, synaptic filamentous actin (F-actin) accumulation, and cytotoxic degranulation, but not granule polarization. Interestingly, we also find that the loss of FLNa augments the target cell-induced expression of IFN-γ and TNF-α by NK cells, correlating with enhanced activation signals such as Ca2+ mobilization, ERK, and NF-κB, and a delayed down-modulation of the NKG2D receptor. Thus, our results identify FLNa as a new regulator of NK cell effector functions during their decision to kill target cells through a balanced regulation of NK cell cytotoxicity vs cytokine production. Moreover, this study implicates the cross-linking/bundling of F-actin mediated by FLNa as a necessary process coordinating optimal NK effector functions.

Secondary Structure and X-Ray Crystallographic Analysis of the Glideosome-Associated Connector (GAC) from Toxoplasma gondii

A model for parasitic motility has been proposed in which parasite filamentous actin (F-actin) is attached to surface adhesins by a large component of the glideosome, known as the glideosome-associated connector protein (GAC). This large 286 kDa protein interacts at the cytoplasmic face of the plasma membrane with the phosphatidic acid-enriched inner leaflet and cytosolic tails of surface adhesins to connect them to the parasite actomyosin system. GAC is observed initially to the conoid at the apical pole and re-localised with the glideosome to the basal pole in gliding parasite. GAC presumably functions in force transmission to surface adhesins in the plasma membrane and not in force generation. Proper connection between F-actin and the adhesins is as important for motility and invasion as motor operation itself. This notion highlights the need for new structural information on GAC interactions, which has eluded the field since its discovery. We have obtained crystals that diffracted to 2.6-2.9 Å for full-length GAC from Toxoplasma gondii in native and selenomethionine-labelled forms. These crystals belong to space group P212121, cell dimensions are roughly a=119 Å, b=123Å, c=221Å, α=90, β=90, γ=90 with 1 molecule per asymmetric unit, suggesting a more compact conformation than previously proposed.

Information flow, Gating, and Energetics in dimeric molecular motors

Molecular motors belonging to the kinesin and myosin super family hydrolyze ATP by cycling through a sequence of chemical states. These cytoplasmic motors are dimers made up of two linked identical monomeric globular proteins. Fueled by the free energy generated by ATP hydrolysis, the motors walk on polar tracks (microtubule or filamentous actin) processively, which means that only one head detaches and executes a mechanical step while the other stays bound to the track. Thus, the one motor head must regulate chemical state of the other, referred to as "gating", a concept that is not fully understood. Inspired by experiments, showing that only a fraction of the energy from ATP hydrolysis is used to advance the kinesin motors against load, we demonstrate that additional energy is used for coordinating the chemical cycles of the two heads in the dimer - a feature that characterizes gating. To this end, we develop a general framework based on information theory and stochastic thermodynamics, and establish that gating could be quantified in terms of information flow between the motor heads. Applications of the theory to kinesin-1 and Myosin V show that information flow occurs, with positive cooperativity, at external resistive loads that are less than a critical value, Fc. When force exceeds Fc, effective information flow ceases. Interestingly, Fc, which is independent of the input energy generated through ATP hydrolysis, coincides with force at which the probability of backward steps starts to increase. Our findings suggest that transport efficiency is optimal only at forces less than Fc, which implies that these motors must operate at low loads under in vivo conditions.

Syk-MyD88 Axis Is a Critical Determinant of Inflammatory-Response in Activated Macrophages

BackgroundInflammation, a vital immune response to infection and injury, is mediated by macrophage activation. While spleen tyrosine kinase (Syk) and myeloid differentiation primary response 88 (MyD88) are reportedly involved in inflammatory responses in macrophages, their roles and underlying mechanisms are largely unknown.MethodsHere, the role of the MyD88-Syk axis and the mechanism by which Syk and MyD88 cooperate during macrophage-mediated inflammatory responses are explored using knockout conditions of these proteins and mutation strategy as well as flowcytometric and immunoblotting analyses.ResultsSyk rapidly activates the nuclear factor-kappa B (NF-κB) signaling pathway in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and the activation of the NF-κB signaling pathway is abolished in Syk−/− RAW264.7 cells. MyD88 activates Syk and Syk-induced activation of NF-κB signaling pathway in LPS-stimulated RAW264.7 cells but Syk-induced inflammatory responses are significantly inhibited in MyD88−/− RAW264.7 cells. MyD88 interacts with Syk through the tyrosine 58 residue (Y58) in the hemi-immunoreceptor tyrosine-based activation motif (ITAM) of MyD88, leading to Syk activation and Syk-induced activation of the NF-κB signaling pathway. Src activates MyD88 by phosphorylation at Y58 via the Src kinase domain. In addition, Ras-related C3 botulinum toxin substrate 1 (Rac1) activation and Rac1-induced formation of filamentous actin (F actin) activate Src in LPS-stimulated RAW264.7 cells.ConclusionsThese results suggest that the MyD88-Syk axis is a critical player in macrophage-mediated inflammatory responses, and its function is promoted by an upstream Src kinase activated by Rac1-generated filamentous actin (F-actin).

Actin Crosslinking Family Protein 7 Deficiency Does Not Impair Hearing in Young Mice

To enable hearing, the sensory hair cell contains specialized subcellular structures at its apical region, including the actin-rich cuticular plate and circumferential band. ACF7 (actin crosslinking family protein 7), encoded by the gene Macf1 (microtubule and actin crosslinking factor 1), is a large cytoskeletal crosslinking protein that interacts with microtubules and filamentous actin to shape cells. ACF7 localizes to the cuticular plate and the circumferential band in the hair cells of vertebrates. The compelling expression pattern of ACF7 in hair cells, combined with conserved roles of this protein in the cytoskeleton of various cell types in invertebrates and vertebrates, led to the hypothesis that ACF7 performs a key function in the subcellular architecture of hair cells. To test the hypothesis, we conditionally target Macf1 in the inner ears of mice. Surprisingly, our data show that in young, but mature, conditional knockout mice cochlear hair cell survival, planar cell polarity, organization of the hair cells within the organ of Corti, and capacity to hear are not significantly impacted. Overall, these results fail to support the hypothesis that ACF7 is an essential hair cell protein in young mice, and the purpose of ACF7 expression in the hair cell remains to be understood.

Correlative cryo-imaging of the cellular universe with soft X-rays and laser light used to track F-actin structures in mammalian cells

Imaging of actin filaments is crucial due to the integral role that they play in many cellular functions such as intracellular transport, membrane remodelling and cell motility. Visualizing actin filaments has so far relied on fluorescence microscopy and electron microscopy/tomography. The former lacks the capacity to capture the overall local ultrastructure, while the latter requires rigorous sample preparation that can lead to potential artefacts, and only delivers relatively small volumes of imaging data at the thinnest areas of a cell. In this work, a correlative approach utilizing in situ super-resolution fluorescence imaging and cryo X-ray tomography was used to image bundles of actin filaments deep inside cells under near-native conditions. In this case, fluorescence 3D imaging localized the actin bundles within the intracellular space, while X-ray tomograms of the same areas provided detailed views of the local ultrastructure. Using this new approach, actin trails connecting vesicles in the perinuclear area and hotspots of actin presence within and around multivesicular bodies were observed. The characteristic prevalence of filamentous actin in cytoplasmic extensions was also documented.

MMP9 Differentially Regulates Proteins Involved in Actin Polymerization and Cell Migration during TGF-β-Induced EMT in the Lens

Fibrotic cataracts have been attributed to transforming growth factor-beta (TGF-β)-induced epithelial-to-mesenchymal transition (EMT). Using mouse knockout (KO) models, our laboratory has identified MMP9 as a crucial protein in the TGF-β-induced EMT process. In this study, we further revealed an absence of alpha-smooth muscle actin (αSMA) and filamentous-actin (F-actin) stress fibers in MMP9KO mouse lens epithelial cell explants (LECs). Expression analysis using NanoString revealed no marked differences in αSMA (ACTA2) and beta-actin (β-actin) (ACTB) mRNA between the lenses of TGF-β-overexpressing (TGF-βtg) mice and TGF-βtg mice on a MMP9KO background. We subsequently conducted a protein array that revealed differential regulation of proteins known to be involved in actin polymerization and cell migration in TGF-β-treated MMP9KO mouse LECs when compared to untreated controls. Immunofluorescence analyses using rat LECs and the novel MMP9-specific inhibitor, JNJ0966, revealed similar differential regulation of cortactin, FAK, LIMK1 and MLC2 as observed in the array. Finally, a reduction in the nuclear localization of MRTF-A, a master regulator of cytoskeletal remodeling during EMT, was observed in rat LECs co-treated with JNJ0966 and TGF-β. In conclusion, MMP9 deficiency results in differential regulation of proteins involved in actin polymerization and cell migration, and this in turn prevents TGF-β-induced EMT in the lens.