The Drosophila Hippo pathway transcription factor Scalloped and its co-factors alter each other's chromatin binding dynamics and transcription in vivo.

The Hippo pathway is an important regulator of organ growth and cell fate. The major mechanism by which Hippo is known to control transcription is by dictating the nucleo-cytoplasmic shuttling rate of Yorkie, a transcription co-activator, which promotes transcription with the DNA binding protein Scalloped. The nuclear biophysical behavior of Yorkie and Scalloped, and whether this is regulated by the Hippo pathway, remains unexplored. Using multiple live-imaging modalities on Drosophila tissues, we found that Scalloped interacts with DNA on a broad range of timescales, and enrichment of Scalloped at sites of active transcription is mediated by longer DNA dwell times. Further, Yorkie increased Scalloped's DNA dwell time, whereas the repressors Nervous fingers 1 (Nerfin-1) and Tondu-domain-containing growth inhibitor (Tgi) decreased it. Therefore, the Hippo pathway influences transcription not only by controlling nuclear abundance of Yorkie but also by modifying the DNA binding kinetics of the transcription factor Scalloped.

Ultralow- and Low-Background Surfaces for Single-Molecule Localization Microscopy of Multistep Biointerfaces for Single-Molecule Sensing.

Single-molecule localization microscopy (SMLM) has created the opportunity of pushing fluorescence microscopy from being a biological imaging tool to a surface characterization and possibly even a quantitative analytical tool. The latter could be achieved by molecular counting using pointillist SMLM data sets. However, SMLM is especially sensitive to background fluorescent signals, which influences any subsequent analysis. Therefore, fabricating sensing surfaces that resist nonspecific adsorption of proteins, even after multiple modification steps, has become paramount. Herein is reported two different ways to modify surfaces: dichlorodimethylsilane-biotinylated bovine serum albumin-Tween-20 (DbT20) and poly-l-lysine grafted polyethylene glycol (PLL-PEG) mixed with biotinylated PLL-PEG (PLL-PEG/PEGbiotin). The results show that the ability to resist nonspecific adsorption of DbT20 surfaces deteriorates with an increase in the number of modification steps required after the addition of the DbT20, which limits the applicability of this surface for SMLM. As such, a new surface for SMLM that employs PLL-PEG/PEGbiotin was developed that exhibits ultralow amounts of nonspecific protein adsorption even after many modification steps. The utility of the surface was demonstrated for human influenza hemagglutinin-tagged mEos2, which was directly pulled down from cell lysates onto the PLL-PEG/PEGbiotin surface. The results strongly indicated that the PLL-PEG/PEGbiotin surface satisfies the criteria of SMLM imaging of a negligible background signal and negligible nonspecific adsorption.

A mobile endocytic network connects clathrin-independent receptor endocytosis to recycling and promotes T cell activation.

Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling.

New Insights into How Trafficking Regulates T Cell Receptor Signaling.

There is emerging evidence that exocytosis plays an important role in regulating T cell receptor (TCR) signaling. The trafficking molecules involved in lytic granule (LG) secretion in cytotoxic T lymphocytes (CTL) have been well-studied due to the immune disorder known as familial hemophagocytic lymphohistiocytosis (FHLH). However, the knowledge of trafficking machineries regulating the exocytosis of receptors and signaling molecules remains quite limited. In this review, we summarize the reported trafficking molecules involved in the transport of the TCR and downstream signaling molecules to the cell surface. By combining this information with the known knowledge of LG exocytosis and general exocytic trafficking machinery, we attempt to draw a more complete picture of how the TCR signaling network and exocytic trafficking matrix are interconnected to facilitate T cell activation. This also highlights how membrane compartmentalization facilitates the spatiotemporal organization of cellular responses that are essential for immune functions.

Palmitoylation is required for intracellular trafficking of influenza B virus NB protein and efficient influenza B virus growth in vitro.

All influenza viruses bud and egress from lipid rafts within the apical plasma membrane of infected epithelial cells. As a result, all components of progeny virions must be transported to these lipid rafts for assembly and budding. Although the mechanism of transport for other influenza proteins has been elucidated, influenza B virus (IBV) glycoprotein NB subcellular localization and transport are not understood completely. To address the aforementioned properties of NB, a series of trafficking experiments were conducted. Here, we showed that NB co-localized with markers specific for the endoplasmic reticulum (ER) and Golgi region. The data from chemical treatment of NB-expressing cells by Brefeldin A, a fungal antibiotic and a known chemical inhibitor of the protein secretory pathway, further confirmed that NB is transported through the ER-Golgi pathway as it restricted NB localization to the perinuclear region. Using NB deletion mutants, the hydrophobic transmembrane domain was identified as being required for NB transport to the plasma membrane. Furthermore, palmitoylation was also required for transport of NB to the plasma membrane. Systematic mutation of cysteines to serines in NB demonstrated that cysteine 49, likely in a palmitoylated form, is also required for transport to the plasma membrane. Surprisingly, further analysis demonstrated that in vitro replication of NBC49S mutant virus was delayed relative to the parental IBV. The results demonstrated that NB is the third influenza virus protein to have been shown to be palmitoylated and together these findings may aid in future studies aimed at elucidating the function of NB.

Biochemical characterization of the small hydrophobic protein of avian metapneumovirus.

Avian metapneumovirus (AMPV) is a paramyxovirus that has three membrane proteins (G, F, and SH). Among them, the SH protein is a small type II integral membrane protein that is incorporated into virions and is only present in certain paramyxoviruses. In the present study, we show that the AMPV SH protein is modified by N-linked glycans and can be released into the extracellular environment. Furthermore, we demonstrate that glycosylated AMPV SH proteins form homodimers through cysteine-mediated disulfide bonds, which has not been reported previously for SH proteins of paramyxoviruses.

Application of a split luciferase complementation assay for the detection of viral protein-protein interactions.

Intraviral protein-protein interactions are critical for virus survival in the host. Discovery of such interactions is important to understand molecular mechanisms of viral replication and pathogenesis. The development of a cell-based assay that can be employed to examine systematically viral protein interactions is described. The method, known as the split luciferase complementation assay (SLCA), is based on the principle that N- and C-terminal domains of luciferase alone do not emit luminescence; however, if fused to interacting proteins the two non-functional halves can be brought into close enough proximity through a specific protein-protein interaction to restore the functions of the enzyme and emit detectable light. The well-studied influenza B polymerase acidic protein (PA) and basic protein 1 (PB1) interaction was used as a model system to develop the assay. Consistent with previous studies, a strong PA-PB1 interaction was demonstrated in the assay. The PA-PB1 interaction was also disrupted by single amino acid mutations in the N-terminal domain of PB1 that is responsible for binding PA. The described SLCA is highly specific and easy to perform, and thus may be useful for studying protein-protein interactions in viral diseases.

Topology and cellular localization of the small hydrophobic protein of avian metapneumovirus.

The small hydrophobic protein (SH) is a type II integral membrane protein that is packaged into virions and is only present in certain paramyxoviruses including metapneumovirus. In addition to a highly divergent primary sequence, SH proteins vary significantly in size amongst the different viruses. Human respiratory syncytial virus (HRSV) encodes the smallest SH protein consisting of only 64 amino acids, while metapneumoviruses have the longest SH protein ranging from 174 to 179 amino acids in length. Little is currently known about the cellular localization and topology of the metapneumovirus SH protein. Here we characterize for the first time metapneumovirus SH protein with respect to topology, subcellular localization, and transport using avian metapneumovirus subgroup C (AMPV-C) as a model system. We show that AMPV-C SH is an integral membrane protein with N(in)C(out) orientation located in both the plasma membrane as well as within intracellular compartments, which is similar to what has been described previously for SH proteins of other paramyxoviruses. Furthermore, we demonstrate that AMPV-C SH protein localizes in the endoplasmic reticulum (ER), Golgi, and cell surface, and is transported through ER-Golgi secretory pathway.

The cellular endosomal sorting complex required for transport pathway is not involved in avian metapneumovirus budding in a virus-like-particle expression system.

Avian metapneumovirus (AMPV) is a paramyxovirus that principally causes respiratory disease and egg production drops in turkeys and chickens. Together with its closely related human metapneumovirus (HMPV), they comprise the genus Metapneumovirus in the family Paramyxoviridae. Little is currently known about the mechanisms involved in the budding of metapneumovirus. By using AMPV as a model system, we showed that the matrix (M) protein by itself was insufficient to form virus-like-particles (VLPs). The incorporation of M into VLPs was shown to occur only when both the viral nucleoprotein (N) and the fusion (F) proteins were co-expressed. Furthermore, we provided evidence indicating that two YSKL and YAGL segments encoded within the M protein were not a functional late domain, and the endosomal sorting complex required for transport (ESCRT) machinery was not involved in metapneumovirus budding, consistent with a recent observation that human respiratory syncytial virus, closely related to HMPV, uses an ESCRT-independent budding mechanism. Taken together, these results suggest that metapneumovirus budding is independent of the ESCRT pathway and the minimal budding machinery described here will aid our future understanding of metapneumovirus assembly and egress.

Nuclear localization of influenza B polymerase proteins and their binary complexes.

The viral RNA-dependent RNA polymerases of influenza A and B are trimeric complexes of PA, PB1, and PB2 subunits that are crucial for both transcription and replication of the viral genome. Unlike the significant progress made recently in understanding nuclear transport and molecular assembly of influenza A polymerase, little is known about the influenza B polymerase, although influenza B viruses cause severe upper respiratory disease in humans. The aim of this study was to characterize nuclear localization of the influenza B RNA polymerase proteins and binary complexes. We demonstrated that each polymerase protein has a nuclear localization function, and among them, the PB2 protein exclusively locates to the nucleus while PA and PB1 proteins are associated with the cytoplasm and the nucleus. Furthermore, we show that pairwise binary complexes are formed among the influenza B subunits (PA-PB1, PA-PB2, and PB1-PB2) and both PB1-PB2 and PA-PB2 complexes are predominantly associated with the nucleus while the PA-PB1 complex exhibits both nuclear and cytoplasmic fluorescence signals. Results of our studies represent the first step toward the understanding of nuclear transport and molecular assembly within the influenza B polymerase complex.