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Exocytosis of large-dense core vesicles in neuroendocrine cells is a highly regulated, calcium-dependent process, mediated by networks of interrelated proteins and lipids. Here, I describe experimental procedures for studies of selective spatial and temporal aspects of exocytosis at the plasma membrane, or in its proximity, using adrenal chromaffin cells. The assay utilizes primary cells subjected to a brief ultrasonic pulse, resulting in the formation of thin, flat inside-out plasma membranes with attached secretory vesicles and elements of cell cytoskeleton. In this model, secretion of plasma membrane-attached secretory vesicles was found to be dependent on calcium and sensitive to clostridial neurotoxins. Depending on the probe selected for secretory vesicle cargo, protein, and/or lipid detection, this simple assay is versatile, fast and inexpensive, and offers excellent spatial resolution.

The plant trans-Golgi network (TGN) is a multifunctional organelle derived from the Golgi. It consists of tubulovesicular compartments scattered in the cytosol. They produce secretory vesicles delivering proteins and polysaccharides to the cell wall. They also serve as early endosomal compartments, receiving endocytic cargos from the plasma membrane. This versatility is thought to originate from functional variations among individual TGN compartments. Correlative light and electron microscopy (CLEM) combines the imaging capability of light microscopy and electron microscopy (EM) to determine the location of macromolecules in EM images in the cellular context. It is possible to identify organelles associated with specific fluorescent markers and examine their membrane architectures at nanometer-level resolutions using CLEM. In this chapter, we will explain the CLEM method that our lab uses to investigate functional and structural heterogeneity among individual TGN compartments in plant cells.

Diverse neuropeptides participate in cell–cell communication to coordinate neuronal and endocrine regulation of physiological processes in health and disease. Neuropeptides are short peptides ranging in length from ~3 to 40 amino acid residues that are involved in biological functions of pain, stress, obesity, hypertension, mental disorders, cancer, and numerous health conditions. The unique neuropeptide sequences define their specific biological actions. Significantly, this review article discusses how the neuropeptide field is at the crest of expanding knowledge gained from mass-spectrometry-based neuropeptidomic studies, combined with proteomic analyses for understanding the biosynthesis of neuropeptidomes. The ongoing expansion in neuropeptide diversity lies in the unbiased and global mass-spectrometry-based approaches for identification and quantitation of peptides. Current mass spectrometry technology allows definition of neuropeptide amino acid sequence structures, profiling of multiple neuropeptides in normal and disease conditions, and quantitative peptide measures in biomarker applications to monitor therapeutic drug efficacies. Complementary proteomic studies of neuropeptide secretory vesicles provide valuable insight into the protein processes utilized for neuropeptide production, storage, and secretion. Furthermore, ongoing research in developing new computational tools will facilitate advancements in mass-spectrometry-based identification of small peptides. Knowledge of the entire repertoire of neuropeptides that regulate physiological systems will provide novel insight into regulatory mechanisms in health, disease, and therapeutics.

Cells synthesize, store, and secrete, on demand, products such as hormones, growth factors, neurotransmitters, or digestive enzymes. Cellular cargos destined for secretion are packaged and stored in membranous sacs or vesicles, which are transported via microtubule and actin railroad systems, to dock and establish continuity at the base of specialized cup-shaped plasma membrane structures called porosomes to release their contents. The fusion of membrane-bound secretory vesicles at the porosome base, and the molecular mechanism of secretory vesicle swelling required for content expulsion during cell secretion, is discussed in this Springer Brief. These new findings in the last 15 years have resulted in a paradigm shift in our understanding of the secretory process in cells and has given birth to a new field in biology—NanoCellBiology.

In this brief review we have attempted to highlight the core principles and mechanism of regulated exocytosis and highlight some of the key questions and opportunities for analysis of the vesicle lifecycle in dendrites. Many questions remain: how are secretory vesicles targeted to dendrites? Does local vesicle biogenesis or recycling occur in dendrites? Do functionally distinct vesicle pools reside in the dendrite? Are secretory vesicles docked at active zones with distinct Ca2+ channels in dendrites? Are SVs and LDCVs differentially targeted and regulated in dendrites? How is vesicle priming and recycling controlled? We are entering a fascinating period in which many key molecular, biochemical, imaging, biophysical and bioinformatics tools can be assembled to allow functional analysis of dendritic release and forge the framework to understand the integrative function of dendritic retrograde signalling in health & disease.