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Interleukin 15 (IL-15) is an essential cytokine for the survival and proliferation of natural killer (NK) cells. IL-15 activates signaling by the β and common γ (γ(c)) chain heterodimer of the IL-2 receptor through trans-presentation by cells expressing IL-15 bound to the α chain of the IL-15 receptor (IL-15Rα). We show here that membrane-associated IL-15Rα–IL-15 complexes are transferred from presenting cells to NK cells through trans-endocytosis and contribute to the phosphorylation of ribosomal protein S6 and NK cell proliferation. NK cell interaction with soluble or surface-bound IL-15Rα–IL-15 complex resulted in Stat5 phosphorylation and NK cell survival at a concentration or density of the complex much lower than required to stimulate S6 phosphorylation. Despite this efficient response, Stat5 phosphorylation was reduced after inhibition of metalloprotease-induced IL-15Rα–IL-15 shedding from trans-presenting cells, whereas S6 phosphorylation was unaffected. Conversely, inhibition of trans-endocytosis by silencing of the small GTPase TC21 or expression of a dominant-negative TC21 reduced S6 phosphorylation but not Stat5 phosphorylation. Thus, trans-endocytosis of membrane-associated IL-15Rα–IL-15 provides a mode of regulating NK cells that is not afforded to IL-2 and is distinct from activation by soluble IL-15. These results may explain the strict IL-15 dependence of NK cells and illustrate how the cellular compartment in which receptor–ligand interaction occurs can influence functional outcome.

The ability of fluorescent proteins (FPs) to fold robustly is fundamental to the autocatalytic formation of the chromophore. While the importance of the tertiary protein structure is well appreciated, the impact of individual amino acid mutations for FPs is often not intuitive and requires direct testing. In this study, we describe the engineering of a monomeric photoswitchable FP, moxMaple3, for use in oxidizing cellular environments, especially the eukaryotic secretory pathway. Surprisingly, a point mutation to replace a cysteine substantially improved the yield of correctly folded FP capable of chromophore formation, regardless of cellular environment. The improved folding of moxMaple3 increases the fraction of visibly tagged fusion proteins, as well as FP performance in PALM super-resolution microscopy, and thus makes moxMaple3 a robust monomeric FP choice for PALM and optical highlighting applications.

Each faculty recruiting season, many postdocs ask, “What is a chalk talk?” The chalk talk is many things—a sales pitch, a teaching demonstration, a barrage of questions, and a description of a future research program. The chalk talk is arguably the most important component of a faculty search interview. Yet few postdocs or grad students receive training or practice in giving a chalk talk. In the following essay, I’ll cover the basics of chalk talk design and preparation.

We have developed a generalized in vitro compartmentalization-based bead display selection strategy that allows for the identification of enzymes that can perform ligation reactions. Although a number of methods have been developed to evolve such enzymes, many of them are limited in library size (10(6) -10(7) ), do not select for enzymes using a scheme that allows for multiple turnover, or only work on enzymes specific to nucleic acids. This approach is amenable to screening libraries of up to 10(12) protein variants by allowing beads to be overloaded with up to 10(4) unique mutants. Using this approach we isolated a variant of sortase A from Staphylococcus aureus that shows a 114-fold enhancement in kcat /KM in the absence of calcium compared to the wild-type and improved resistance to the inhibitory effects of cell lysates. Unlike the wild-type protein, the newly selected variant shows intracellular activity in the cytoplasm of eukaryotic cells where it may prove useful for intracellular labeling or synthetic biological applications. Biotechnol. Bioeng. 2016;113: 1647-1657. © 2016 Wiley Periodicals, Inc.

Foxp3+CD4+ regulatory T (Treg) cells are essential for preventing fatal autoimmunity and safeguard immune homeostasis in vivo. While expression of the transcription factor Foxp3 and IL-2 signals are both required for the development and function of Treg cells, the commitment to the Treg cell lineage occurs during thymic selection upon T cell receptor (TCR) triggering, and precedes the expression of Foxp3. Whether signals beside TCR contribute to establish Treg cell epigenetic and functional identity is still unknown. Here, using a mouse model with reduced IL-2 signaling, we show that IL-2 regulates the positioning of the pioneer factor SATB1 in CD4+ thymocytes and controls genome wide chromatin accessibility of thymic-derived Treg cells. We also show that Treg cells receiving only low IL-2 signals can suppress endogenous but not WT autoreactive T cell responses in vitro and in vivo. Our findings have broad implications for potential therapeutic strategies to reprogram Treg cells in vivo., Regulatory T (Treg) cells are developed in the thymus, and are essential for suppressing detrimental autoimmunity. Here the authors show, using mice with dampened interleukin 2 (IL-2) signaling, that IL-2 helps position the pioneer factor SATB1 to control genome-wide chromatin accessibility to facilitate Treg cell lineage commitment in the thymus.

The saga of fluorescence recovery after photobleaching (FRAP) illustrates how disparate technical developments impact science. Starting with the classic 1976 Axelrod et al. work in Biophysical Journal, FRAP (originally fluorescence photobleaching recovery) opened the door to extraction of quantitative information from photobleaching experiments, laying the experimental and theoretical groundwork for quantifying both the mobility and the mobile fraction of a labeled population of proteins. Over the ensuing years, FRAP's reach dramatically expanded, with new developments in GFP technology and turn-key confocal microscopy, which enabled measurement of protein diffusion and binding/dissociation rates in virtually every compartment within the cell. The FRAP technique and data catalyzed an exchange of ideas between biophysicists studying membrane dynamics, cell biologists focused on intracellular dynamics, and systems biologists modeling the dynamics of cell activity. The outcome transformed the field of cellular biology, leading to a fundamental rethinking of long-held theories of cellular dynamism. Here, we review the pivotal FRAP studies that made these developments and conceptual changes possible, which gave rise to current models of complex cell dynamics.

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs.

Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.

Fluorescent proteins (FPs) that can be optically highlighted enable PALM super-resolution microscopy and pulse-chase experiments of cellular molecules. Most FPs evolved in cytoplasmic environments either in the original source organism or in the cytoplasm of bacteria during the course of optimization for research applications. Consequently, many FPs may fold incorrectly in the chemically distinct environments in subcellular organelles. Here, we describe the first monomeric photoswitchable (from green to bright red) FP adapted for oxidizing environments.

Pathogenic New World hemorrhagic fever mammarenaviruses (NWM) utilize Glycoprotein 1 (GP1) to target the apical domain of the human transferrin receptor (hTfR) for facilitating cell entry. However, the conservation between their GP1s is low. Considering this and the slow evolutionary progression of mammals compared to viruses, therapeutic targeting of hTfR provides an attractive avenue for cross-strain inhibition and diminishing the likelihood of escape mutants. Aptamers present unique advantages for the development of inhibitors to vial entry, including ease of synthesis, lack of immunogenicity, and potentially cold-chain breaking solutions to diseases endemic to South America. Here, recognizing that in vivo competition with the natural ligand, transferrin (Tf), likely drove the evolution of GP1 to recognize the apical domain, we performed competitive in vitro selections against hTfR-expressing cells with supplemented Tf. The resultant minimized aptamer, Waz, binds the apical domain of the receptor and inhibits infection of human cells by recombinant NWM in culture (EC50 ≃400 nmol/l). Aptamer multimerization further enhanced inhibition >10-fold (EC50 ≃30 nmol/l). Together, our results highlight the ability to use a competitor to bias the outcome of a selection and demonstrate how avidity effects can be leveraged to enhance both aptamer binding and the potency of viral inhibition.

We have developed a generalized in vitro compartmentalization-based bead display selection strategy that allows for the identification of enzymes that can perform ligation reactions. Although a number of methods have been developed to evolve such enzymes, many of them are limited in library size (10(6) -10(7) ), do not select for enzymes using a scheme that allows for multiple turnover, or only work on enzymes specific to nucleic acids. This approach is amenable to screening libraries of up to 10(12) protein variants by allowing beads to be overloaded with up to 10(4) unique mutants. Using this approach we isolated a variant of sortase A from Staphylococcus aureus that shows a 114-fold enhancement in kcat /KM in the absence of calcium compared to the wild-type and improved resistance to the inhibitory effects of cell lysates. Unlike the wild-type protein, the newly selected variant shows intracellular activity in the cytoplasm of eukaryotic cells where it may prove useful for intracellular labeling or synthetic biological applications. Biotechnol. Bioeng. 2016;113: 1647-1657. © 2016 Wiley Periodicals, Inc.

Gap junctions (GJs) are made up of plaques of laterally clustered intercellular channels and the membranes in which the channels are embedded. Arrangement of channels within a plaque determines subcellular distribution of connexin binding partners and sites of intercellular signaling. Here, we report the discovery that some connexin types form plaque structures with strikingly different degrees of fluidity in the arrangement of the GJ channel subcomponents of the GJ plaque. We uncovered this property of GJs by applying fluorescence recovery after photobleaching to GJs formed from connexins fused with fluorescent protein tags. We found that connexin 26 (Cx26) and Cx30 GJs readily diffuse within the plaque structures, whereas Cx43 GJs remain persistently immobile for more than 2 min after bleaching. The cytoplasmic C terminus of Cx43 was required for stability of Cx43 plaque arrangement. We provide evidence that these qualitative differences in GJ arrangement stability reflect endogenous characteristics, with the caveat that the sizes of the GJs examined were necessarily large for these measurements. We also uncovered an unrecognized effect of non-monomerized fluorescent protein on the dynamically arranged GJs and the organization of plaques composed of multiple connexin types. Together, these findings redefine our understanding of the GJ plaque structure and should be considered in future studies using fluorescent protein tags to probe dynamics of highly ordered protein complexes.

Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses.

To perform quantitative live cell imaging, investigators require fluorescent reporters that accurately report protein localization and levels, while minimally perturbing the cell. Yet, within the biochemically distinct environments of cellular organelles, popular fluorescent proteins (FPs), including EGFP, can be unreliable for quantitative imaging, resulting in the underestimation of protein levels and incorrect localization. Specifically, within the secretory pathway, significant populations of FPs misfold and fail to fluoresce due to non-native disulphide bond formation. Furthermore, transmembrane FP-fusion constructs can disrupt organelle architecture due to oligomerizing tendencies of numerous common FPs. Here, we describe a powerful set of bright and inert FPs optimized for use in multiple cellular compartments, especially oxidizing environments and biological membranes. Also, we provide new insights into the use of red FPs in the secretory pathway. Our monomeric ‘oxFPs’ finally resolve long-standing, underappreciated and important problems of cell biology and should be useful for a number of applications. Quantitative live cell imaging of protein trafficking suffers from misfolding and inappropriate disulphide bond formation of fluorescent proteins in the secretory pathway. Here, the authors present an optimized collection of fluorescent proteins suitable for use in oxidizing subcellular compartments.