Of the Orthomyxoviridae family of viruses, only influenza A viruses are thought to exist as multiple subtypes and has non-human maintenance hosts. In April 2011, nasal swabs were collected for virus isolation from pigs exhibiting influenza-like illness. Subsequent electron microscopic, biochemical, genetic studies identified an orthomyxovirus with seven RNA segments approximately 50% overall amino acid identity human C virus. Based on its organizational similarities this been provisionally...

The small G proteins Cdc42, Rac1, and Rac2 regulate the rearrangements of actin membrane necessary for Fcgamma receptor-mediated phagocytosis by macrophages. Activated, GTP-bound bind to p21-binding domain (PBD) PAK1, this interaction provided a basis microscopic methods localize activation these inside cells. Fluorescence resonance energy transfer-based stoichiometry fluorescent chimeras actin, PBD, was used quantify protein relative movements during IgG-opsonized erythrocytes. dynamics...

Macropinosomes are large endocytic vesicles that form in ruffling regions of plasma membrane. To analyze signal organization relative to ruffle closure into circular ruffles and cup macropinosomes, this study used quantitative microscopy measure 3′ phosphoinositides small-GTPase activities a representative subset forming macropinosomes. Macropinocytosis was stimulated by the addition macrophage colony-stimulating factor (M-CSF) macrophages expressing fluorescent reporter proteins....

Kinesin motor proteins drive the transport of cellular cargoes along microtubule tracks. How protein activity is controlled in cells unresolved, but it likely coupled to changes conformation and cargo association. By applying quantitative method fluorescence resonance energy transfer (FRET) stoichiometry fluorescent (FP)-labeled kinesin heavy chain (KHC) light (KLC) subunits live cells, we studied overall structural organization Kinesin-1 active inactive states. Inactive molecules are folded...

Clathrin-mediated endocytosis (CME) internalizes plasma membrane by reshaping small regions of the cell surface into spherical vesicles. The key mechanistic question how coat assembly produces curvature has been studied with molecular and cellular structural biology approaches, without direct visualization process in living cells; resulting two competing models for bending. Here we use polarized total internal reflection fluorescence microscopy (pol-TIRF) combined electron, atomic force,...

Many studies of endocytosis and phagocytosis presume that organelles containing a single kind internalized particle exhibit invariant patterns protein phospholipid association as they mature inside cells. To test this presumption, fluorescent chimeras were expressed in RAW 264.7 macrophages, time-lapse ratiometric fluorescence microscopy was used to measure the maturation dynamics individual phagosomes IgG-opsonized erythrocytes. Quantitative analysis revealed consistent for YFP β-actin,...

Listeria monocytogenes (Lm) evade microbicidal defences inside macrophages by secreting a pore-forming cytolysin listeriolysin O (LLO), which allows Lm to escape vacuoles. LLO also inhibits vacuole fusion with lysosomes, indicates alters chemistry prior release of into cytoplasm. Using fluorescent probes measure membrane permeability, calcium and pH, we identified small perforations in vacuoles containing wild-type but not LLO-deficient (hly-) Lm. The released molecules persisted for several...

Fcgamma receptor (FcgammaR)-mediated phagocytosis of IgG-coated particles is regulated by 3'-phosphoinositides (3'PIs) and several classes small GTPases, including ARF6 from the ADP Ribosylation Factor subfamily. The insensitivity to brefeldin A (BFA), an inhibitor certain ARF guanine nucleotide exchange factors (GEFs), previously indicated that ARF1 did not participate in phagocytosis. In this study, we show was activated during FcgammaR-mediated blocking normal cycling inhibited phagosome...

Macroautophagy involves lysosomal/vacuolar elimination of long-lived proteins and entire organelles from the cytosol. The process begins with formation a double-membrane vesicle that sequesters bulk cytoplasm, or specific cargo destined for delivery. completed fuses lysosome/vacuole limiting membrane, releasing its content into organelle lumen subsequent degradation recycling resulting macromolecules. A majority autophagy-related (Atg) are required at step formation. integral membrane...

During Fcγ receptor (FcR)-mediated phagocytosis by macrophages, cytoplasm advances over IgG-coated particles the sequential ligation of FcR in plasma membranes. If signaling was strictly autonomous, then signals generated during should be proportional to number ligated receptors. By measuring FcR-dependent responses beads coated with various densities IgG, this study identified nonlinear that organizes an all or none response particle ingestion. Phagocytosis IgG at low density either stalled...

Abstract The identification of transcription factor binding sites and cis-regulatory motifs is a frontier whereupon the rules governing protein–DNA are being revealed. Here, we developed new method (DEep Sequence Shape mOtif or DESSO) for motif prediction using deep neural networks binomial distribution model. DESSO outperformed existing tools, including DeepBind, in predicting 690 human ENCODE ChIP-sequencing datasets. Furthermore, deep-learning framework expanded discovery beyond...

Fcgamma Receptor (FcR)-mediated phagocytosis by macrophages requires phosphatidylinositol 3-kinase (PI3K) and activation of the Rho-family GTPases Cdc42 Rac1. is activated at advancing edge phagocytic cup, where actin concentrated, deactivated base cup. The timing 3' phosphoinositide (3'PI) concentration changes in cup membranes suggests a role for 3'PIs deactivation Cdc42. This study examined relationships between PI3K patterns GTPase signaling during phagosome formation. Inhibition...

ABSTRACT The human immunodeficiency virus type 1 structural polyprotein Pr55 Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated importance capsid C-terminal domain (CA-CTD), nucleocapsid (NC), membrane association in Gag-Gag interactions, but relationships between these factors remain unclear. In this study, we systematically altered CA-CTD, NC, ability to bind determine relative contributions of, interplay between,...

Clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis in mammalian cells. Acute depletion of cholesterol disrupts CME, motivating analysis CME dynamics the context human disorders metabolism. We report that inhibition post-squalene biosynthesis impairs CME. Imaging bending pit ultrastructure reveals prolonged clathrin lifetimes shallow clathrin-coated structures, suggesting progressive impairment curvature generation...

Two mutations (K141E, K141N) in the small heat shock protein (sHSP) HSP22 (HSPB8) are associated with inherited peripheral motor neuron disorders distal hereditary neuropathy type II and axonal Charcot-Marie-Tooth disease 2L. is known to form homodimers, heterodimers other sHSPs, larger oligomers. In an effort elucidate cellular basis for these diseases, we have determined ability of mutant interact itself, wild-type HSP22, sHSPs that abundant neurons. Using yeast two-hybrid method,...

Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET was extended imaging of multiple donor-acceptor pairs, thereby enabling visualization biochemical events within single living cell. These methods require numerous equations that must be defined on case-by-case basis. Here, we present universal multispectral method...

Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure dynamics of macropinosome formation relative PI3K activity. We show that multiple ruffling morphologies produce macropinosomes majority form through collisions adjacent PI3K-rich ruffles. By combining...

Abstract Microbes in soil navigate interactions by recognizing kin, forming social groups, exhibiting antagonistic behavior, and engaging competitive kin rivalry. Here, we investigated a novel phenomenon of self-growth suppression (sibling rivalry) observed Bradyrhizobium diazoefficiens USDA 110. Swimming colonies 110 developed distinct demarcation line inter-colony zone when inoculated adjacent to each other. In addition self, suppressed growth other strains several bacteria. We...

Abstract Macrophage differentiation, phenotype, and function have been assessed extensively in vitro by predominantly deriving human macrophages from peripheral blood. It is accepted that there are differences between isolated different tissues; however, the importance of anatomical source for differentiation characterization less clear. Here, phenotype were evaluated derived bone marrow or Macrophages differentiated adherence heterogenous cell populations CD14 isolation polarized with IFNγ...

Three mutations (R120G, Q151X, and 464delCT) in the small heat shock protein alphaB-crystallin cause inherited myofibrillar myopathy. In an effort to elucidate molecular basis for associated myopathy, we have determined following these mutant proteins: (i) formation of aggregates transfected cells; (ii) partition into different subcellular fractions; (iii) phosphorylation status; (iv) ability interact with themselves, wild-typealphaB-crystallin, other proteins that are abundant muscles. We...