The human blood-brain barrier (BBB) comprises a single layer of brain microvascular endothelial cells (HBMECs) protecting the brain from bloodborne pathogens. Meningitis is among the most serious diseases, but the mechanisms by which major meningitis-causing bacterial pathogens cross the BBB to reach the brain remain poorly understood. We found that Streptococcus pneumoniae, group B Streptococcus, and neonatal meningitis Escherichia coli commonly exploit a unique vesicle fusion mechanism to hitchhike on transferrin receptor (TfR) transcytosis to cross the BBB and illustrated the details of this process in human BBB model in vitro and mouse model. Toll-like receptor signals emanating from bacteria-containing vesicles (BCVs) trigger K33-linked polyubiquitination at Lys168 and Lys181 of the innate immune regulator TRAF3 and then activate the formation of a protein complex containing the guanine nucleotide exchange factor RCC2, the small GTPase RalA and exocyst subcomplex I (SC I) on BCVs. The distinct function of SEC6 in SC I, interacting directly with RalA on BCVs and the SNARE protein SNAP23 on TfR vesicles, tethers these two vesicles and initiates the fusion. Our results reveal that innate immunity triggers a unique modification of TRAF3 and the formation of the HBMEC-specific protein complex on BCVs to authenticate the precise recognition and selection of TfR vesicles to fuse with and facilitate bacterial penetration of the BBB.

Fungi are an important group of microorganisms that play crucial roles in a variety of ecological and biotechnological processes. Fungi depend on intracellular protein trafficking, which involves moving proteins from their site of synthesis to the final destination within or outside the cell. The soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are vital components of vesicle trafficking and membrane fusion, ultimately leading to the release of cargos to the target destination. The v-SNARE (vesicle-associated SNARE) Snc1 is responsible for anterograde and retrograde vesicle trafficking between the plasma membrane (PM) and Golgi. It allows for the fusion of exocytic vesicles to the PM and the subsequent recycling of Golgi-localized proteins back to the Golgi via three distinct and parallel recycling pathways. This recycling process requires several components, including a phospholipid flippase (Drs2-Cdc50), an F-box protein (Rcy1), a sorting nexin (Snx4-Atg20), a retromer submit, and the COPI coat complex. Snc1 interacts with exocytic SNAREs (Sso1/2, Sec9) and the exocytic complex to complete the process of exocytosis. It also interacts with endocytic SNAREs (Tlg1 and Tlg2) during endocytic trafficking. Snc1 has been extensively investigated in fungi and has been found to play crucial roles in various aspects of intracellular protein trafficking. When Snc1 is overexpressed alone or in combination with some key secretory components, it results in enhanced protein production. This article will cover the role of Snc1 in the anterograde and retrograde trafficking of fungi and its interactions with other proteins for efficient cellular transportation.

Artificial cells are based on dynamic compartmentalized systems. Thus, remodeling of membrane-bound systems, such as giant unilamellar vesicles, is finding applications beyond biological studies, to engineer cell-mimicking structures. Giant unilamellar vesicle fusion is rapidly becoming an essential experimental step as artificial cells gain prominence in synthetic biology. Several techniques have been developed to accomplish this step, with varying efficiency and selectivity. To date, characterization of vesicle fusion has relied on small samples of giant vesicles, examined either manually or by fluorometric assays on suspensions of small and large unilamellar vesicles. Automation of the detection and characterization of fusion products is now necessary for the screening and optimization of these fusion protocols. To this end, we implemented a fusion assay based on fluorophore colocalization on the membranes and in the lumen of vesicles. Fluorescence colocalization was evaluated within single compartments by image segmentation with minimal user input, allowing the application of the technique to high-throughput screenings. After detection, statistical information on vesicle fluorescence and morphological properties can be summarized and visualized, assessing lipid and content transfer for each object by the correlation coefficient of different fluorescence channels. Using this tool, we report and characterize the unexpected fusogenic activity of sodium chloride on phosphatidylcholine giant vesicles. Lipid transfer in most of the vesicles could be detected after 20 h of incubation, while content exchange only occurred with additional stimuli in around 8% of vesicles.

Some of the most conspicuous aging phenotypes of C. elegans are related to post-reproductive production of vitellogenins (Vtg), which form yolk protein (YP) complexes after processing and lipid loading. Vtg/YP levels show huge increases with age, and inhibition of this extends lifespan, but how subcellular and organism-wide distribution of these proteins changes with age has not been systematically explored. Here, this has been done to understand how vitellogenesis promotes aging. The age-associated changes of intestinal vitellogenin vesicles (VVs), pseudocoelomic yolk patches (PYPs), and gonadal yolk organelles (YOs) have been characterized by immuno-electron microscopy. We find that from reproductive adult day 2 (AD 2) to post-reproductive AD 6 and AD 9, intestinal VVs expand from 0.2 to 3-4 μm in diameter or by >3000 times in volume, PYPs increase by >3 times in YP concentration and volume, while YOs in oocytes shrink slightly from 0.5 to 0.4 μm in diameter or by 49% in volume. In AD 6 and AD 9 worms, mislocalized YOs found in the hypodermis, uterine cells, and the somatic gonadal sheath can reach a size of 10 μm across in the former two tissues. This remarkable size increase of VVs and that of mislocalized YOs in post-reproductive worms are accompanied by extensive fusion between these Vtg/YP-containing vesicular structures in somatic cells. In contrast, no fusion is seen between YOs in oocytes. We propose that in addition to the continued production of Vtg, excessive fusion between VVs and mislocalized YOs in the soma worsen the aging pathologies seen in C. elegans.

Ca2+-triggered neurotransmitter release involves complex regulatory mechanisms, including a series of protein-protein interactions. Three proteins, synaptobrevin (VAMP), synaptosomal-associated protein of 25kDa (SNAP-25) and syntaxin, constitute the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complex that plays key roles in controlling vesicle fusion and exocytosis. Many other proteins participate in the regulation of the processes via direct and/or indirect interaction with the SNARE complex. Although much effort has been made, the regulatory mechanism for exocytosis is still not completely clear. Accumulated evidence indicates that the small GTPase Rab3 and synaptotagmin proteins play important regulatory roles during vesicle fusion and neurotransmitter release. This review outlines our present understanding of the two regulatory proteins, with the focus on the interaction of Rab3 with synaptotagmin in the regulatory process.

Intracellular vesicle trafficking is the fundamental process to maintain the homeostasis of membrane-enclosed organelles in eukaryotic cells. These organelles transport cargo from the donor membrane to the target membrane through the cargo containing vesicles. Vesicle trafficking pathway includes vesicle formation from the donor membrane, vesicle transport, and vesicle fusion with the target membrane. Coat protein mediated vesicle formation is a delicate membrane budding process for cargo molecules selection and package into vesicle carriers. Vesicle transport is a dynamic and specific process for the cargo containing vesicles translocation from the donor membrane to the target membrane. This process requires a group of conserved proteins such as Rab GTPases, motor adaptors, and motor proteins to ensure vesicle transport along cytoskeletal track. Soluble N-ethyl-maleimide-sensitive factor (NSF) attachment protein receptors (SNARE)-mediated vesicle fusion is the final process for vesicle unloading the cargo molecules at the target membrane. To ensure vesicle fusion occurring at a defined position and time pattern in eukaryotic cell, multiple fusogenic proteins, such as synaptotagmin (Syt), complexin (Cpx), Munc13, Munc18 and other tethering factors, cooperate together to precisely regulate the process of vesicle fusion. Dysfunctions of the fusogenic proteins in SNARE-mediated vesicle fusion are closely related to many diseases. Recent studies have suggested that stimulated membrane fusion can be manipulated pharmacologically via disruption the interface between the SNARE complex and Ca2+ sensor protein. Here, we summarize recent insights into the molecular mechanisms of vesicle trafficking, and implications for the development of new therapeutics based on the manipulation of vesicle fusion.

Vesicle fusion is of crucial importance to neuronal communication at neuron terminals. The exquisite but complex fusion machinery for neurotransmitter release is tightly controlled and regulated by protein/neurotransmitter-membrane interactions. Computational \'microscopies\', in particular molecular dynamics simulations and related techniques, have provided notable insight into the physiological process over the past decades, and have made enormous contributions to fields such as neurology, pharmacology and pathophysiology. Here we review the computational advances of protein/neurotransmitter-membrane interactions related to presynaptic vesicle-membrane fusion and neurotransmitter release, and outline the in silico challenges ahead for understanding this important physiological process.

Total internal reflection fluorescence microscopy (TIRFM) provides extremely thin optical sectioning with excellent signal-to-noise ratios, which allows for visualization of membrane dynamics at the cell surface with superb spatiotemporal resolution. In this chapter, TIRFM is used to record and analyze exocytosis of single glucose transporter-4 (GLUT4) containing vesicles in 3T3-L1 adipocytes.

The GLUT4 vesicle fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and a variety of regulatory proteins. For example, synip and tomosyn negatively regulate GLUT4 SNARE-mediated membrane fusion. Here we describe in vitro reconstituted assays to determine the molecular mechanisms of SNAREs, synip, and tomosyn. These methods can also be extended to the studies of other types of membrane fusion events.

Whole-cell patch clamping is a standard method to monitor the secretion of synaptic vesicles. In this chapter, we describe the basic steps of whole-cell patch clamping for measuring synaptic exocytosis, aiming to provide reference for researchers who are new to this field.