The endoplasmic reticulum (ER) consists of a polygonal network of sheets and tubules interconnected by three-way junctions. This network undergoes continual remodeling through competing processes: the branching and fusion of tubules forms new three-way junctions and new polygons, and junction sliding and ring closure leads to polygon loss. However, little is known about the machinery required to generate and maintain junctions. We previously reported that yeast Lnp1 localizes to ER junctions, and that loss of Lnp1 leads to a collapsed, densely reticulated ER network. In mammalian cells, only approximately half the junctions contain Lnp1. Here we use live cell imaging to show that mammalian Lnp1 (mLnp1) affects ER junction mobility and hence network dynamics. Three-way junctions with mLnp1 are less mobile than junctions without mLnp1. Newly formed junctions that acquire mLnp1 remain stable within the ER network, whereas nascent junctions that fail to acquire mLnp1 undergo rapid ring closure. These findings imply that mLnp1 plays a key role in stabilizing nascent three-way ER junctions.

Nanomaterials possess distinctive physicochemical properties (e.g., small sizes, high surface area-to-volume ratios) and promise a wide variety of applications, ranging from design of high quality consumer products to effective disease diagnosis and therapy. These properties can lead to toxic effects, potentially hindering advance in nanotechnology. In this study, we have synthesized and characterized purified and stable (non-aggregation) silver nanoparticles (Ag NPs, 41.6±9.1 nm in average diameters), and utilized early-developing (cleavage-stage) zebrafish embryos (critical aquatic and eco- species) as in vivo model organisms to probe diffusion and toxicity of Ag NPs. We found that single Ag NPs (30–72 nm diameters) passively diffused into the embryos through chorionic pores via random Brownian motion and stayed inside the embryos throughout their entire development (120 hours-post-fertilization, hpf). Dose and size dependent toxic effects of the NPs on embryonic development were observed, showing the possibility of tuning biocompatibility and toxicity of the NPs. At lower concentrations of the NPs (≤ 0.02 nM), 75–91% of embryos developed to normal zebrafish. At the higher concentrations of NPs (≥ 0.20 nM), 100% of embryos became dead. At the concentrations in between (0.02–0.2 nM), embryos developed to various deformed zebrafish. Number and sizes of individual Ag NPs embedded in tissues of normal and deformed zebrafish at 120 hpf were quantitatively analyzed, showing deformed zebrafish with higher number of larger NPs than normal zebrafish, and size-dependent nanotoxicity. By comparing with our previous studies of smaller Ag NPs (11.6±3.5 nm), the results further demonstrate striking size-dependent nanotoxicity that, at the same molar concentration, the larger Ag NPs (41.6±9.1 nm) are more toxic than the smaller Ag NPs (11.6±3.5 nm).

Multidrug membrane transporters (efflux pumps) in both prokaryotes and eukaryotes are responsible for impossible treatments of a wide variety of diseases, including infections and cancer, underscoring the importance of better understanding their structures and functions for design of effective therapies. In this study, we designed and synthesized two silver nanoparticle (Ag NP) solution with average diameters of 13.1 ± 2.5 nm (8.1–38.6 nm) and 91.0 ± 9.3 nm (56–120 nm), and used the size-dependent plasmonic spectra of single NPs to probe the size-dependent transport kinetics of MexAB-OprM (multidrug transporter) in Pseudomonas (P.) aeruginosa in real-time at nanometer resolution. We found that the accumulation of intracellular NPs in wild-type (WT) cells was higher than in nalB1 (over-expression of MexAB-OprM), but less than ΔABM (deletion of MexAB-OprM). In the presence of proton ionophores (CCCP, inhibitor of proton-motive-force), we found that intracellular NPs in nalB1 were nearly doubled. These results suggest that the MexAB-OprM is responsible for the extrusion of NPs out of cells and NPs (orders of magnitude larger than conventional antibiotics) are the substrates of the transporter, which indicates that the substrates may trigger the assembly of the efflux pump optimized for the extrusion of the encountered substrates. We found that the smaller NPs stayed longer inside the cells than larger NPs, suggesting the size-dependent efflux kinetics of the cells. This study shows that multi-sized NPs can be used to mimic various sizes of antibiotics for probing the size-dependent efflux kinetics of multidrug membrane transporters in single living cells.

Nanomaterials exhibit distinctive physicochemical properties and promise a wide range of applications from nanotechnology to nanomedicine, which raise serious concerns about their potential environmental impacts on ecosystems. Unlike any conventional chemicals, nanomaterials are highly heterogeneous, and their properties can alter over time. These unique characteristics underscore the importance of study of their properties and effects on living organisms in real time at single nanoparticle (NP) resolution. Here we report the development of single-NP plasmonic microscopy and spectroscopy (dark-field optical microscopy and spectroscopy, DFOMS) and ultrasensitive in vivo assay (cleavage-stage zebrafish embryos, critical aquatic species) to study transport and toxicity of single silver nanoparticles (Ag NPs, 95.4 ± 16.0 nm) on embryonic developments. We synthesized and characterized purified and stable (non-aggregation) Ag NPs, determined their sizes and doses (number), and their transport mechanisms and effects on embryonic development in vivo in real time at single-NP resolution. We found that single Ag NPs passively entered the embryos through their chorionic pores via random Brownian diffusion and stayed inside the embryos throughout their entire development (120 h), suggesting that the embryos can bio-concentrate trace NPs from their environment. Our studies show that higher doses and larger sizes of Ag NPs cause higher toxic effects on embryonic development, demonstrating that the embryos can serve as ultrasensitive in vivo assays to screen biocompatibility and toxicity of the NPs and monitor their potential release into aquatic ecosystems.

Background:Atlastin is large GTPase that catalyzes the homotypic fusion of ER membranes. Results: In vitro and in vivo studies reveal that the C-terminal tail of Atlastin affects its function. Conclusion:The amphipathic C-terminal tail of Atlastin destabilizes lipid bilayers to promote membrane fusion. Significance: Describing the mechanism of Atlastin-mediated fusion is a critical step in our understanding of ER structure formation.