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Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin‐2 mutations, p.A618T, and p.S619L, impair Ca2+‐induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high‐temporal resolution techniques, such as patch‐clamp capacitance measurements and carbon‐fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin‐2 mutants inhibits a dynamin and F‐actin‐dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin‐2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)‐labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1‐dimethyl‐4‐phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca2+‐induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)‐linked p.A618T and p.S619L mutations in dynamin‐2 affect exocytosis and endocytosis, being the disruption of F‐actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations. [ABSTRACT FROM AUTHOR]

Neuropeptides are vital for cell-cell communication and function in the regulation of the nervous and endocrine systems. They are generated by post-translational modification (PTM) steps resulting in small active peptides generated from prohormone precursors. Phosphorylation is a significant PTM for the bioactivity of neuropeptides. From the known diversity of distinct neuropeptide functions, it is hypothesized that the extent of phosphorylation varies among different neuropeptides. To assess this hypothesis, neuropeptide-containing dense core secretory vesicles from bovine adrenal medullary chromaffin cells were subjected to global phosphopeptidomics analyses by liquid chromatography (LC)-mass spectrometry (MS/MS). Phosphopeptides were identified directly by LC-MS/MS and indirectly by phosphatase treatment followed by LC-MS/MS. The data identified numerous phosphorylated peptides derived from neuropeptide precursors such as chromogranins, secretogranins, proenkephalin and pro-NPY. Phosphosite occupancies were observed at high and low levels among identified peptides and many of the high occupancy phosphopeptides represent prohormone-derived peptides with currently unknown bioactivities. Peptide sequence analyses demonstrated SxE as the most prevalent phosphorylation site motif, corresponding to phosphorylation sites of the Fam20C protein kinase known to be present in the secretory pathway. The range of high to low phosphosite occupancies for neuropeptides demonstrates cellular regulation of neuropeptide phosphorylation. Graphical Abstract ᅟ.

The importance of the immediately releasable pool (IRP) of vesicles was proposed to reside in the maintenance of chromaffin cell secretion during the firing of action potentials at basal physiological frequencies. To accomplish this duty, IRP should be replenished as a function of time. We have previously reported that an action potential‐like stimulus (APls) triggers the release of ~50% IRP, followed by a fast dynamin‐dependent endocytosis and an associated rapid replenishment process. In this work, we investigated the endocytosis and IRP replenishment produced after the exocytosis of variable IRP fractions in mice primary chromaffin cell cultures. Exocytosis and endocytosis were estimated by membrane capacitance measurements obtained in patch‐clamped cells. In addition to the dynamin‐dependent fast endocytosis activated after the application of APls or 5 ms squared depolarizations, we found that depolarizations lasting 25–50 ms, which release >80% of IRP, are related with a fast dynamin‐independent, Ca2+‐ and protein kinase C (PKC)‐dependent endocytosis (time constant <1 s). PKC inhibitors, such as staurosporine, bisindolylmaleimide XI, PKC 19–31 peptide, and prolonged treatments with high concentrations of phorbol esters, reduced and decelerated this endocytosis. Additionally, we found that the inhibition of PKC also abolished a slow component of replenishment (time constant ~8 s) observed after total IRP exocytosis. Therefore, our results suggest that PKC contributes to the coordination of membrane retrieval and vesicle replenishment mechanisms that occur after the complete exocytosis of IRP. [ABSTRACT FROM AUTHOR]

Sjögren’s syndrome (SjS) is a chronic autoimmune disease characterized by immune cell infiltration of the exocrine glands, mainly the salivary and lacrimal glands. Despite recent advances in the clinical and mechanistic characterization of the disease, its etiology remains largely unknown. Here, we report that mice with a deficiency for either Atg7 or Atg3, which are enzymes involved in the ubiquitin modification pathway, in the salivary glands exhibit a SjS-like phenotype, characterized by immune cell infiltration with autoantibody detection, acinar cell death, and dry mouth. Prior to the onset of the SjS-like phenotype in these null mice, we detected an accumulation of secretory vesicles in the acinar cells of the salivary glands and found that GATE16, an uncharacterized autophagy-related molecule activated by ATG7 (E1-like enzyme) and ATG3 (E2-like enzyme), was highly expressed in these cells. Notably, GATE16 was activated by isoproterenol, an exocytosis inducer, and localized on the secretory vesicles in the acinar cells of the salivary glands. Failure to activate GATE16 was correlated with exocytosis defects in the acinar cells of the salivary glands in Atg7 and Atg3 cKO mice. Taken together, our results show that GATE16 activation regulated by the autophagic machinery is crucial for exocytosis and that defects in this pathway cause SjS. [ABSTRACT FROM AUTHOR]

Exocytosis is a universal process of eukaryotic cells, consisting of fusion between the vesicle and the plasma membranes, leading to the formation of a fusion pore, a channel through which vesicle cargo exits into the extracellular space. In 1986, Rand and Parsegian proposed several stages to explain the nature of membrane fusion. Following stimulation, it starts with focused stress destabilization of membranes in contact, followed by the coalescence of two membrane surfaces. In the next fraction of a millisecond, restabilization of fused membranes is considered to occur to maintain the cell’s integrity. This view predicted that once a fusion pore is formed, it must widen abruptly, irreversibly and fully, whereby the vesicle membrane completely integrates with and collapses into the plasma membrane (full fusion exocytosis). However, recent experimental evidence has revealed that once the fusion pore opens, it may also reversibly close (transient or kiss-and-run exocytosis). Here, we present a historical perspective on understanding the mechanisms that initiate the membrane merger and fusion pore formation. Next, post-fusion mechanisms that regulate fusion pore stability are considered, reflecting the state in which the forces of widening and constriction of fusion pores are balanced. Although the mechanisms generating these forces are unclear, they may involve lipids and proteins, including SNAREs, which play a role not only in the pre-fusion but also post-fusion stages of exocytosis. How molecules stabilize the fusion pore in the open state is key for a better understanding of fusion pore physiology in health and disease.

RAB GTPases are major determinants of membrane identity that have been exploited as highly specific reporters to study intracellular traffic in vivo. A score of fungal papers have considered individual RABs, but systematic, integrated studies on the localization and physiological role of these regulators and their effectors have been performed only with Aspergillus nidulans. These studies have influenced the intracellular trafficking field beyond fungal specialists, leading to findings such as the maturation of trans‐Golgi (TGN) cisternae into post‐Golgi RAB11 secretory vesicles, the concept that these RAB11 secretory carriers are loaded with three molecular nanomotors, the understanding of the role of endocytic recycling mediated by RAB6 and RAB11 in determining the hyphal mode of life, the discovery that early endosome maturation and the ESCRT pathway are essential, the identification of specific adaptors of dynein‐dynactin to RAB5 endosomes, the exquisite dependence that autophagy displays on RAB1 activity, the role of TRAPPII as a GEF for RAB11, or the conclusion that the RAB1‐to‐RAB11 transition is not mediated by TRAPP maturation. A remarkable finding was that the A. nidulans Spitzenkörper contains four RABs: RAB11, Sec4, RAB6, and RAB1. How these RABs cooperate during exocytosis represents an as yet outstanding question. [ABSTRACT FROM AUTHOR]

Impaired insulin release is a hallmark of type 2 diabetes and is closely related to chronically elevated glucose concentrations, known as “glucotoxicity.” However, the molecular mechanisms by which glucotoxicity impairs insulin secretion remain poorly understood. In addition to known kiss-and-run and kiss-and-stay fusion events in INS-1 cells, ultrafast Hessian structured illumination microscopy (Hessian SIM) enables full fusion to be categorized according to the newly identified structures, such as ring fusion (those with enlarged pores) or dot fusion (those without apparent pores). In addition, we identified four fusion intermediates during insulin exocytosis: initial pore opening, vesicle collapse, enlarged pore formation, and final pore dilation. Long-term incubation in supraphysiological doses of glucose reduced exocytosis in general and increased the occurrence of kiss-and-run events at the expense of reduced full fusion. In addition, hyperglycemia delayed pore opening, vesicle collapse, and enlarged pore formation in full fusion events. It also reduced the size of apparently enlarged pores, all of which contributed to the compromised insulin secretion. These phenotypes were mostly due to the hyperglycemia-induced reduction in syntaxin-1A (Stx-1A) and SNAP-25 protein, since they could be recapitulated by the knockdown of endogenous Stx-1A and SNAP-25. These findings suggest essential roles for the vesicle fusion type and intermediates in regulating insulin secretion from pancreatic beta cells in normal and disease conditions.

The maintenance of the secretory response requires a continuous replenishment of releasable vesicles. It was proposed that the immediately releasable pool (IRP) is important in chromaffin cell secretion during action potentials applied at basal physiological frequencies, because of the proximity of IRP vesicles to voltage‐dependent Ca2+ channels. However, previous reports showed that IRP replenishment after depletion is too slow to manage such a situation. In this work, we used patch‐clamp measurements of membrane capacitance, confocal imaging of F‐actin distribution, and cytosolic Ca2+ measurements with Fura‐2 to re‐analyze this problem in primary cultures of mouse chromaffin cells. We provide evidence that IRP replenishment has one slow (time constant between 5 and 10 s) and one rapid component (time constant between 0.5 and 1.5 s) linked to a dynamin‐dependent fast endocytosis. Both, the fast endocytosis and the rapid replenishment component were eliminated when 500 nM Ca2+ was added to the internal solution during patch‐clamp experiments, but they became dominant and accelerated when the cytosolic Ca2+ buffer capacity was increased. In addition, both rapid replenishment and fast endocytosis were retarded when cortical F‐actin cytoskeleton was disrupted with cytochalasin D. Finally, in permeabilized chromaffin cells stained with rhodamine‐phalloidin, the cortical F‐actin density was reduced when the Ca2+ concentration was increased in a range of 10–1000 nM. We conclude that low cytosolic Ca2+ concentrations, which favor cortical F‐actin stabilization, allow the activation of a fast endocytosis mechanism linked to a rapid replenishment component of IRP. [ABSTRACT FROM AUTHOR]

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.

Summary: The plant Golgi apparatus (sensu lato: Golgi stack + Trans Golgi Network, TGN) is a highly polar and mobile key organelle lying at the junction of the secretory and endocytic pathways. Unlike its counterpart in animal cells it does not disassemble during mitosis. It modifies glycoproteins sent to it from the endoplasmic reticulum (ER), it recycles ER resident proteins, it sorts proteins destined for the vacuole from secretory proteins, it receives proteins internalised from the plasma membrane and either recycles them to the plasma membrane or retargets them to the vacuole for degradation. In functional terms the Golgi apparatus can be likened to a car factory, with incoming (COPII traffic) and returning (COPI traffic) railway lines at the entry gate, and a distribution centre (the TGN) at the exit gate of the assembly hall. In the assembly hall we have a conveyor belt system where the incoming car parts are initially assembled (in the cis‐area) then gradually modified into different models (processing of secretory cargo) as the cars pass along the production line (cisternal maturation). After being released the trans‐area, the cars (secretory cargos) are moved out of the assembly hall and passed on to the distribution centre (TGN), where the various models are placed onto different trains (cargo sorting into carrier vesicles) for transport to the car dealers. Cars with motor problems are returned to the factory for repairs (endocytosis to the TGN). This simple analogy also incorporates features of quality control at the COPII entry gate with defective parts being returned to the manufacturing center (the ER) via the COPI trains (vesicles). In recent years, numerous studies have contributed to our knowledge on Golgi function and structure in both animals, yeast and plants. This review, rather than giving a balanced account of the structure as well as of the function of the Golgi apparatus has purposely a marked slant towards plant Golgi ultrastructure integrating findings from the mammalian/animal field. [ABSTRACT FROM AUTHOR]

In this paper, we present a mathematical model to describe filamentous fungal growth based on intracellular secretory vesicles (SVs), which transport cell wall components to the hyphal tip. Vesicular transport inside elongating hyphae is modeled as an advection–diffusion–reaction equation with a moving boundary, transformed into fixed coordinates, and discretized using a high‐order weighted essentially nonoscillatory discretization scheme. The model describes the production and the consumption of SVs with kinetic functions. Simulations are subsequently compared against distributions of SVs visualized by enhanced green fluorescent protein in young Aspergillus niger hyphae after germination. Intensity profile data are obtained using an algorithm scripted in ImageJ that extracts mean intensity distributions from 3D time‐lapse confocal measurement data. Simulated length growth is in good agreement with the experimental data. Our simulations further show that a decrease of effective vesicle transport velocity towards the tip can explain the observed tip accumulation of SVs. [ABSTRACT FROM AUTHOR]

Premature intrapancreatic trypsinogen activation is widely regarded as an initiating event for acute pancreatitis. Previous studies have alternatively implicated secretory vesicles, endosomes, lysosomes, or autophagosomes/autophagolysosomes as the primary site of trypsinogen activation, from which a cell-damaging proteolytic cascade originates. To identify the subcellular compartment of initial trypsinogen activation we performed a time-resolution analysis of the first 12 h of caerulein-induced pancreatitis in transgenic light chain 3 (LC3)-GFP autophagy reporter mice. Intrapancreatic trypsin activity increased within 60 min and serum amylase within 2 h, but fluorescent autophagosome formation only by 4 h of pancreatitis in parallel with a shift from cytosolic LC3-I to membranous LC3-II on Western blots. At 60 min, activated trypsin in heavier subcellular fractions was co-distributed with cathepsin B, but not with the autophagy markers LC3 or autophagy protein 16 (ATG16). Supramaximal caerulein stimulation of primary pancreatic acini derived from LC3-GFP mice revealed that trypsinogen activation is independent of autophagolysosome formation already during the first 15 min of exposure to caerulein. Co-localization studies (with GFP-LC3 autophagosomes versus Ile–Pro–Arg–AMC trypsin activity and immunogold-labelling of lysosomal-associated membrane protein 2 [LAMP-2] versus trypsinogen activation peptide [TAP]) indicated active trypsin in autophagolysosomes only at the later timepoints. In conclusion, during the initiating phase of caerulein-induced pancreatitis, premature protease activation develops independently of autophagolysosome formation and in vesicles arising from the secretory pathway. However, autophagy is likely to regulate overall intracellular trypsin activity during the later stages of this disease. [ABSTRACT FROM AUTHOR]

Aim: It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by Ca2+ entry through voltage‐dependent Ca2+ channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca2+ current and intracellular Ca2+ increase. In this work we investigated if such a mechanism contributes to secretory vesicle exocytosis in mouse chromaffin cells. Methods: Exocytosis was evaluated by patch‐clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic Ca2+ was estimated using epifluorescence microscopy and fluo‐8 (salt form). Results: Cells stimulated by brief depolatizations in absence of extracellular Ca+2 show moderate but consistent exocytosis, even in presence of high cytosolic BAPTA concentration and pharmacological inhibition of Ca+2 release from intracellular stores. This exocytosis is tightly dependent on membrane potential, is inhibited by neurotoxin Bont‐B (cleaves the v‐SNARE synaptobrevin), is very fast (saturates with time constant <10 ms), it is followed by a fast endocytosis sensitive to the application of an anti‐dynamin monoclonal antibody, and recovers after depletion in <5 s. Finally, this exocytosis was inhibited by: (i) ω‐agatoxin IVA (blocks P/Q‐type Ca2+ channel gating), (ii) in cells from knock‐out P/Q‐type Ca2+ channel mice, and (iii) transfection of free synprint peptide (interferes in P/Q channel‐exocytic proteins association). Conclusion: We demonstrated that Ca2+‐independent and voltage‐dependent exocytosis is present in chromaffin cells. This process is tightly coupled to membrane depolarization, and is able to support secretion during action potentials at low basal rates. P/Q‐type Ca2+ channels can operate as voltage sensors of this process. [ABSTRACT FROM AUTHOR]

• Vti1a and Vti1b are canonical endolysosomal Qb-SNAREs involved in neuronal secretion. • The role of Vti proteins in secretion can be explained by a Vti -dependent availability of secretory organelles and proteins of the secretion machinery at exocytic sites. • Vti proteins regulate Golgi export of secretory molecules, probably by retrieving sorting molecules to this organelle. Vti proteins are conserved from yeast to humans and regulate intracellular membrane trafficking by providing one specific SNARE domain, the Qb SNARE, to the four helical SNARE bundle that drives membrane fusion. Two mammalian Vti genes, Vti1a and Vti1b are reported to regulate distinct aspects of endolysosomal trafficking and retrograde transport to the Golgi, but have also been implicated in synaptic vesicle secretion. In this review, we summarize the current evidence for the role of Vti proteins in intracellular trafficking in different cells. We propose that, despite some unique aspects, the two mammalian VTI genes have largely redundant functions in neurosecretory cells and recycle molecules required for the sorting of regulated cargo to the Golgi. Defects in this recycling also lead to defects in synaptic transmission and dense core vesicle secretion. [ABSTRACT FROM AUTHOR]

Over the last years, there is accumulating evidence that acidic organelles can accumulate and release Ca2+ upon cell activation. Hence, reliable recording of Ca2+ dynamics in these compartments is essential for understanding the physiopathological aspects of acidic organelles. Genetically encoded Ca2+ indicators (GECIs) are valuable tools to monitor Ca2+ in specific locations, although their use in acidic compartments is challenging due to the pH sensitivity of most available fluorescent GECIs. By contrast, bioluminescent GECIs have a combination of features (marginal pH sensitivity, low background, no phototoxicity, no photobleaching, high dynamic range and tunable affinity) that render them advantageous to achieve an enhanced signal-to-noise ratio in acidic compartments. This article reviews the use of bioluminescent aequorin-based GECIs targeted to acidic compartments. A need for more measurements in highly acidic compartments is identified. • Acidic organelles are high Ca2+ content stores that can take up and release Ca2+ upon cell activation. • The optimal Ca2+ indicator for acidic organelles should be pH resistant and of low Ca2+ affinity. • Aequorin probes have been engineered for monitoring Ca2+ in several mildly acidic organelles across kingdoms of life. • Ca2+ measurements in more acidic compartments are limited. [ABSTRACT FROM AUTHOR]