Project description:Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal "actin hotspots" along axons-spaced ?3-4 µm apart-where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons-a phenomenon we call "actin trails." Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal "actin rings" described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin-but not Arp2/3-dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable "actin rings" providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles.
Project description:The proteins that make up the actin cytoskeleton can self-assemble into a variety of structures. In vitro experiments and coarse-grained simulations have shown that the actin crosslinking proteins α-actinin and fascin segregate into distinct domains in single actin bundles with a molecular size-dependent competition-based mechanism. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that physical confinement can cause these proteins to form much more complex structures, including rings and asters at GUV peripheries and centers; the prevalence of different structures depends on GUV size. Strikingly, we found that α-actinin and fascin self-sort into separate domains in the aster structures with actin bundles whose apparent stiffness depends on the ratio of the relative concentrations of α-actinin and fascin. The observed boundary-imposed effect on protein sorting may be a general mechanism for creating emergent structures in biopolymer networks with multiple crosslinkers.
Project description:Facial action units (AUs) relate to specific local facial regions. Recent efforts in automated AU detection have focused on learning the facial patch representations to detect specific AUs. These efforts have encountered three hurdles. First, they implicitly assume that facial patches are robust to head rotation; yet non-frontal rotation is common. Second, mappings between AUs and patches are defined a priori, which ignores co-occurrences among AUs. And third, the dynamics of AUs are either ignored or modeled sequentially rather than simultaneously as in human perception. Inspired by recent advances in human perception, we propose a dynamic patch-attentive deep network, called D-PAttNet, for AU detection that (i) controls for 3D head and face rotation, (ii) learns mappings of patches to AUs, and (iii) models spatiotemporal dynamics. D-PAttNet approach significantly improves upon existing state of the art.
Project description:Objective- During atherosclerosis, LDLs (low-density lipoproteins) accumulate in the arteries, where they become modified, aggregated, and retained. Such deposits of aggregated LDL (agLDL) can be recognized by macrophages, which attempt to digest and clear them. AgLDL catabolism promotes internalization of cholesterol and foam cell formation, which leads to the progression of atherosclerosis. Therapeutic blockade of this process may delay disease progression. When macrophages interact with agLDL in vitro, they form a novel extracellular, hydrolytic compartment-the lysosomal synapse (LS)-aided by local actin polymerization to digest agLDL. Here, we investigated the specific regulators involved in actin polymerization during the formation of the LS. Approach and Results- We demonstrate in vivo that atherosclerotic plaque macrophages contacting agLDL deposits polymerize actin and form a compartment strikingly similar to those made in vitro. Live cell imaging revealed that macrophage cortical F-actin depolymerization is required for actin polymerization to support the formation of the LS. This depolymerization is cofilin-1 dependent. Using siRNA-mediated silencing, pharmacological inhibition, genetic knockout, and stable overexpression, we elucidate key roles for Cdc42 Rho GTPase and GEF (guanine nucleotide exchange factor) Vav in promoting actin polymerization during the formation of the LS and exclude a role for Rac1. Conclusions- These results highlight critical roles for dynamic macrophage F-actin rearrangement and polymerization via cofilin-1, Vav, and Cdc42 in LS formation, catabolism of agLDL, and foam cell formation. These proteins might represent therapeutic targets to treat atherosclerotic disease.
Project description:BackgroundActin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments.Methodology/principal findingsIn this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore.Conclusions/significanceLifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin's role in tip growing plant cells.
Project description:Cadherins are transmembrane adhesion proteins required for the formation of cohesive tissues.1-4 Intracellular interactions of E-cadherin with the Catenin family proteins, α- and β-catenin, facilitate connections with the cortical actomyosin network. This is necessary for maintaining the integrity of cell-cell adhesion in epithelial tissues.5-11 The supra-molecular architecture of E-cadherin is an important feature of its adhesion function; cis and trans interactions of E-cadherin are deployed12-15 to form clusters, both in cis and trans.11,16-21 Studies in Drosophila embryo have also shown that Drosophila E-cadherin (dE-cad) is organized as finite-sized dynamic clusters that localize with actin patches at cell-cell junctions, in continuous exchange with the extra-junctional pool of dE-cad surrounding the clusters.11,19 Here, we use the ectopic expression of dE-cad in larval hemocytes, which lack endogenous dE-cad to recapitulate functional cell-cell junctions in a convenient model system. We find that, while dE-cad at cell-cell junctions in hemocytes exhibits a clustered trans-paired organization similar to that reported previously in embryonic epithelial tissue, extra-junctional dE-cad is also organized as relatively immobile nanoclusters as well as more loosely packed diffusive oligomers. Oligomers are promoted by cis interactions of the ectodomain, and their growth is counteracted by the activity of cortical actomyosin. Oligomers in turn promote assembly of dense nanoclusters that require cortical actomyosin activity. Thus, cortical actin activity remodels oligomers and generates nanoclusters. The requirement for dynamic actin in the organization of dE-cad at the nanoscale may provide a mechanism to dynamically tune junctional strength.
Project description:Using the "face inversion effect", a hallmark of face perception, we examined network mechanisms supporting face representation by tracking functional magnetic resonance imaging (fMRI) stimulus-dependent dynamic functional connectivity within and between brain networks associated with the processing of upright and inverted faces. We developed a novel approach adapting the general linear model (GLM) framework classically used for univariate fMRI analysis to capture stimulus-dependent fMRI dynamic connectivity of the face network. We show that under the face inversion manipulation, the face and non-face networks have complementary roles that are evident in their stimulus-dependent dynamic connectivity patterns as assessed by network decomposition into components or communities. Moreover, we show that connectivity patterns are associated with the behavioral face inversion effect. Thus, we establish "a network-level signature" of the face inversion effect and demonstrate how a simple physical transformation of the face stimulus induces a dramatic functional reorganization across related brain networks. Finally, we suggest that the dynamic GLM network analysis approach, developed here for the face network, provides a general framework for modeling the dynamics of blocked stimulus-dependent connectivity experimental designs and hence can be applied to a host of neuroimaging studies.
Project description:Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s(-1), respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays.
Project description:Force-producing convergence (narrowing) and extension (lengthening) of tissues by active intercalation of cells along the axis of convergence play a major role in axial morphogenesis during embryo development in both vertebrates and invertebrates, and failure of these processes in human embryos leads to defects including spina bifida and anencephaly. Here we use Xenopus laevis, a system in which the polarized cell motility that drives this active cell intercalation has been related to the development of forces that close the blastopore and elongate the body axis, to examine the role of myosin IIB in convergence and extension. We find that myosin IIB is localized in the cortex of intercalating cells, and show by morpholino knockdown that this myosin isoform is essential for the maintenance of a stereotypical, cortical actin cytoskeleton as visualized with time-lapse fluorescent confocal microscopy. We show that this actin network consists of foci or nodes connected by cables and is polarized relative to the embryonic axis, preferentially cyclically shortening and lengthening parallel to the axis of cell polarization, elongation and intercalation, and also parallel to the axis of convergence forces during gastrulation. Depletion of MHC-B results in disruption of this polarized cytoskeleton, loss of the polarized protrusive activity characteristic of intercalating cells, eventual loss of cell-cell and cell-matrix adhesion, and dose-dependent failure of blastopore closure, arguably because of failure to develop convergence forces parallel to the myosin IIB-dependent dynamics of the actin cytoskeleton. These findings bridge the gap between a molecular-scale motor protein and tissue-scale embryonic morphogenesis.
Project description:Glioblastoma (GB) is the most representative form of primary malignant brain tumour. Several studies indicated a pleiotropic role of CXCL8 in cancer due to its ability to modulate the tumour microenvironment, growth and aggressiveness of tumour cell. Previous studies indicated that CXCL8 by its receptors (CXCR1 and CXCR2) induced activation of the PI3K/p-Akt pathway, a crucial event in the regulation of cytoskeleton rearrangement and cell mobilization. Human GB primary cell culture and U-87MG cell line were used to study the effects of CXCR1 and CXCR2 blockage, by a dual allosteric antagonist, on cell migration and cytoskeletal dynamics. The data obtained point towards a specific effect of autocrine CXCL8 signalling on GB cell invasiveness by the activation of pathways involved in cell migration and cytoskeletal dynamics, such as PI3K/p-Akt/p-FAK, p-cortactin, RhoA, Cdc42, Acetylated ?-tubulin and MMP2. All the data obtained support the concept that autocrine CXCL8 signalling plays a key role in the activation of an aggressive phenotype in primary glioblastoma cells and U-87MG cell line. These results provide new insights about the potential of a pharmacological approach targeting CXCR1/CXCR2 pathways to decrease migration and invasion of GB cells in the brain parenchyma, one of the principal mechanisms of recurrence.