Chimeric Newcastle Disease Virus-like Particles Containing DC-Binding Peptide-Fused Haemagglutinin Protect Chickens from Virulent Newcastle Disease Virus and H9N2 Avian Influenza Virus Challenge.

Newcastle disease virus (NDV) and H9N2 subtype Avian influenza virus (AIV) are two notorious avian respiratory pathogens that cause great losses in the poultry industry. Current inactivated commercial vaccines against NDV and AIV have the disadvantages of inadequate mucosal responses, while an attenuated live vaccine bears the risk of mutation. Dendritic cell (DC) targeting strategies are attractive for their potent mucosal and adaptive immune-stimulating ability against respiratory pathogens. In this study, DC-binding peptide (DCpep)-decorated chimeric virus-like particles (cVLPs), containing NDV haemagglutinin-neuraminidase (HN) and AIV haemagglutinin (HA), were developed as a DC-targeting mucosal vaccine candidate. DCpep-decorated cVLPs activated DCs in vitro, and induced potent immune stimulation in chickens, with enhanced secretory immunoglobulin A (sIgA) secretion and splenic T cell differentiation. 40 µg cVLPs can provide full protection against the challenge with homologous, heterologous NDV strains, and AIV H9N2. In addition, DCpep-decorated cVLPs could induce a better immune response when administered intranasally than intramuscularly, as indicated by robust sIgA secretion and a reduced virus shedding period. Taken together, this chimeric VLPs are a promising vaccine candidate to control NDV and AIV H9N2 and a useful platform bearing multivalent antigens.

Visualization of reticulophagy in living cells using an endoplasmic reticulum-targeted p62 mutant.

Reticulophagy is a type of selective autophagy in which protein aggregate-containing and/or damaged endoplasmic reticulum (ER) fragments are engulfed for lysosomal degradation, which is important for ER homeostasis. Several chemical drugs and mutant proteins that promote protein aggregate formation within the ER lumen can efficiently induce reticulophagy in mammalian cells. However, the exact mechanism and cellular localization of reticulophagy remain unclear. In this report, we took advantage of the self-oligomerization property of p62/SQSTM1, an adaptor for selective autophagy, and developed a novel reticulophagy system based on an ER-targeted p62 mutant to investigate the process of reticulophagy in living cells. LC3 conversion analysis via western blot suggested that p62 mutant aggregate-induced ER stress triggered a cellular autophagic response. Confocal imaging showed that in cells with moderate aggregation conditions, the aggregates of ER-targeted p62 mutants were efficiently sequestered by autophagosomes, which was characterized by colocalization with the autophagosome precursor marker ATG16L1, the omegasome marker DFCP1, and the late autophagosomal marker LC3/GATE-16. Moreover, time-lapse imaging data demonstrated that the LC3- or DFCP1-positive protein aggregates are tightly associated with the reticular structures of the ER, thereby suggesting that reticulophagy occurs at the ER and that omegasomes may be involved in this process.

A general strategy for developing cell-permeable photo-modulatable organic fluorescent probes for live-cell super-resolution imaging.

Single-molecule localization microscopy (SMLM) achieves super-resolution imaging beyond the diffraction limit but critically relies on the use of photo-modulatable fluorescent probes. Here we report a general strategy for constructing cell-permeable photo-modulatable organic fluorescent probes for live-cell SMLM by exploiting the remarkable cytosolic delivery ability of a cell-penetrating peptide (rR)3R2. We develop photo-modulatable organic fluorescent probes consisting of a (rR)3R2 peptide coupled to a cell-impermeable organic fluorophore and a recognition unit. Our results indicate that these organic probes are not only cell permeable but can also specifically and directly label endogenous targeted proteins. Using the probes, we obtain super-resolution images of lysosomes and endogenous F-actin under physiological conditions. We resolve the dynamics of F-actin with 10 s temporal resolution in live cells and discern fine F-actin structures with diameters of ~80 nm. These results open up new avenues in the design of fluorescent probes for live-cell super-resolution imaging.

Protein profiling of active cysteine cathepsins in living cells using an activity-based probe containing a cell-penetrating peptide.

Cell-permeable activity-based probes (ABPs) are capable of labeling target proteins in living cells, thereby providing a powerful tool for profiling active enzymes in their native environment. In this study, we describe the synthesis and use of a novel trifunctional cell-permeable activity-based probe (TCpABP) for proteomic profiling of active cysteine cathepsins in living cells. We demonstrate that although TCpABP contains cell-impermeable tags, it was able to enter living cells efficiently via the delivery of a cell-penetrating peptide. TCpABP also allowed simultaneous detection and affinity isolation of labeled proteins with a fluorophore and a biotin motif, respectively. We optimized the enrichment protocol to minimize contaminants and identified 7 cathepsins, 2 of which have never been identified using existing ABPs. We also used a label-free quantification approach to quantify the relative abundances of active cathepsins and compared them with their previously published mRNA expression levels. A high degree of correlation between the mRNA expression levels and protein relative activities was observed for most of the identified cathepsins except cathepsin H. The results herein indicate that TCpABP is valuable for the detection of active cathepsins in living cells and provides useful guidelines for designing novel cell-permeable ABPs for in vivo labeling and their applications in in vivo proteomics studies.

Localization-based super-resolution microscopy with an sCMOS camera.

In the community of localization-based super-resolution microscopy (or called localization microscopy), it is generally believed that the emission of single molecules is so weak that an EMCCD (electron multiplying charge coupled device) camera is necessary to be used as the detector by eliminating read noise. Here we evaluate the possibility of a new kind of low light detector, scientific complementary metal-oxide-semiconductor (sCMOS) camera in localization microscopy. We demonstrate experimentally that sCMOS is capable of imaging actin bundles with FWHM diameter of 37 nm, evidencing the capability of sCMOS in localization microscopy. We further characterize the noise performance of sCMOS and find out that, with the use of a bright fluorescence probe such as d2EosFP, localization microscopy imaging is now working in the shot noise limited region.

Fiber-optic system for monitoring fast photoactivation dynamics of optical highlighter fluorescent proteins.

Characterizing the photoactivation performance of optical highlighter fluorescent proteins is crucial to the realization of photoactivation localization microscopy. In contrast to those fluorescence-based approaches that require complex data processing and calibration procedures, here we report a simple and quantitative alternative, which relies on the measurement of small absorption spectra changes over time with a fiber-optic system. Using Dronpa as a representative highlighter protein, we have investigated the capacity of this system in monitoring the fast photoactivation process.

A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions.

Fluorescent protein (FP) has enabled the analysis of biomolecular interactions in living cells, and bimolecular fluorescence complementation (BiFC) represents one of the newly developed imaging technologies to directly visualize protein-protein interactions in living cells. Although 10 different FPs that cover a broad range of spectra have been demonstrated to support BiFC, only Cerulean (cyan FP variant), Citrine and Venus (yellow FP variants)-based BiFC systems can be used under 37 degrees C physiological temperature. The sensitivity of two mRFP-based red BiFC systems to higher temperatures (i.e., 37 degrees C) limits their applications in most mammalian cell-based studies. Here we report that mLumin, a newly isolated far-red fluorescent protein variant of mKate with an emission maximum of 621 nm, enables BiFC analysis of protein-protein interactions at 37 degrees C in living mammalian cells. Furthermore, the combination of mLumin with Cerulean- and Venus-based BiFC systems allows for simultaneous visualization of three pairs of protein-protein interactions in the same cell. The mLumin-based BiFC system will facilitate simultaneous visualization of multiple protein-protein interactions in living cells and offer the potential to visualize protein-protein interactions in living animals.