ISCCB 2017 - Speaker Program
19th International Symposium on Chromaffin Cell Biology
22nd - 26th August

Tuesday 22nd August

Session number
15:00 Plenary 1 Andrew Peden

Elucidating the role of the qbc SNARE, SNAP29 in post golgi trafficking.

Over the past twenty years, our understanding of the molecular machinery that drives intracellular transport and vesicle fusion has dramatically increased. For the majority of pathways, we now have a good understanding of which SNAREs are involved. However, it is less clear how post-Golgi SNAREs are trafficked to the correct compartments within the cell. My lab, in collaboration with others, has used a combination of biochemical approaches, trafficking assays and structural biology to obtain a mechanistic understanding of how R-SNAREs are packaged into transport vesicles. More recently, my lab has focused on elucidating how SNAREs lacking transmembrane domains are targeted to membranes. In particular, we are characterising the trafficking and function of the poorly characterised Q-SNAREs, SNAP29 and STX19. In my talk, I will discuss the mechanism involved in targeting SNAP29 and STX19 to tubular recycling endosomes and discuss the role of SNAP29 in post-Golgi trafficking.

16:00 Session 1 Bazbek Davletov

Novel pharmacological regulators of exocytosis

Neuronal SNARE proteins form a tight four-helical bundle to allow calcium-triggered fusion of secretory vesicles with the plasma membrane. We are exploring SNARE proteins as drug targets to allow precise modulation of secretion. We previously demonstrated that sphingosine, a sphingolipid metabolite, promotes formation of the SNARE complex required for membrane fusion and also increases the rate of vesicle exocytosis. Recently a fungi-derived sphingosine homologue, FTY720, has been approved for treatment of multiple sclerosis. Considering close structural similarity of sphingosine and FTY720 we investigated whether FTY720 has an effect on regulated exocytosis. Our data demonstrate that FTY720 can activate vesicular synaptobrevin for SNARE complex formation and enhance exocytosis in neuroendocrine cells and neurons. In addition, we are developing new technologies for delivery of biomedicinal enzymes into neurons. We are using a SNARE-based ‘protein stapling’ technique which potentially allows conjugation of therapeutic enzymes to cell-targeting agents. I will describe our recent progress to generate precise neuronal regulators which utilise botulinum neurotoxin SNARE proteases.

Darios FD, Jorgacevski J, Flašker A, Zorec R, García-Martinez V, Villanueva J, Gutiérrez LM, Leese C, Bal M, Nosyreva E, Kavalali ET & Davletov B (2017) Sphingomimetic multiple sclerosis drug FTY720 activates vesicular synaptobrevin and augments neuroendocrine secretion. Scientific Reports, 7.
Mangione AS, Obara I, Maiarú M, Geranton SM, Tassorelli C, Ferrari E, Leese C, Davletov B & Hunt SP (2016) Nonparalytic botulinum molecules for the control of pain. PAIN 157(5): 1045-1055.
Rust A, Leese C, Binz T & Davletov B (2016) Botulinum neurotoxin type C protease induces apoptosis in differentiated human neuroblastoma cells. Oncotarget 7(22):33220-8.

16:30 Session 1 Thomas Binz

Botulinum neurotoxin serotypes, subtypes, and mutants as tools in cell biology and for medical applications

Botulinum neurotoxins comprise seven serologically distinguishable extremely potent toxins (BoNT/A to G). They enter nerve cells via endocytosis subsequent to binding gangliosides and synaptotagmin or SV2. Their catalytic domain (light chain, LC) acts after delivery in the cytosol as a zinc-endoprotease. BoNTs cleave specific members of the three SNARE protein families: VAMP/synaptobrevin, SNAP-25, or syntaxin. SNARE proteolysis inhibits fusion of synaptic vesicles with the presynaptic membrane at the motoneuronal junction and thereby causes botulism. However, for 25 years, BoNTs have served as effective pharmaceutical for the treatment of clinical conditions caused by hyperactivity of cholinergic nerve terminals. The range of their applications today includes neurons that innervate smooth muscles and glands as in overactive bladder and axillary hyperhidrosis. Further expansion of their application to therapeutically relevant hypersecretion of non-neuronal cells like mast cells and macrophages is prevented, as BoNTs do not readily cleave pertinent SNAREs like TI-VAMP/VAMP-7 and SNAP-23. Hence, re-engineering the known LCs is required in order that they can serve in diseases like chronic obstructive pulmonary disease, asthma etc. We attempted to create LC/A with novel properties based on in-depth analysis of its substrate specificity and establishment of a model for its interaction with the virtually non-cleavable human SNAP-23. The results were exploited for re-engineering selected binding pockets in order to adapt the protease to human SNAP-23. To date, LC/A mutants obtained exhibit up to ten-fold increased activity to human SNAP-23. Combinations of individual mutants led to multiple mutants with yet higher activity to human SNAP-23. The large number of recently detected new BoNT subtypes, to date >40, represents a reservoir for LC variants with potentially novel biochemical properties. Characterization of their substrate specificity is underway in various laboratories and might yield BoNTs exploitable in therapy and as new tool for cell biology studies.

17:00 Break
17:20 Session 1 Jernej Jorgaceyski

The anatomy and exocytic properties of astrocytic vesicles

An important mode of intercellular communication involves the release of molecules from cells by exocytosis. Exocytotic transmitter release is most likely regulated by the SNARE complex, which contains a vesicular protein, synaptobrevin2 (Sb2). In this presentation our anatomical study of single vesicles in astrocytes, using super-resolution stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM) will be presented first. Smaller vesicles with diameters of ~65 nm contain amino acid and peptidergic transmitters and larger vesicles with diameters of ~230 nm contain ATP. By using photo bleaching experiments and fluorescence intensity analysis, we determined that an average astrocytic vesicle contains 15–25 endogenous Sb2 molecules. The density of Sb2 molecules appeared to be lower in larger vesicles. Moreover, the distribution of Sb2 molecules in vesicles is non-uniform and one YSpH molecule appeared necessary for fusion pore formation. In the second part of the lecture, the interaction of a single vesicle with the plasma membrane, monitored by a high-resolution membrane capacitance approach, will be presented. To determine unitary exocytotic event properties, reflecting the interaction of a single vesicle with the plasma membrane, discrete increases in membrane capacitance, revealed that astrocyte stimulation increases the frequency of predominantly transient fusion exocytotic events in smaller vesicles, whereas larger vesicles proceed more likely to full fusion exocytosis. To determine whether this reflects a lower density of SNARE proteins in larger vesicles, we treated astrocytes with botulinum neurotoxins D and E, which reduced exocytotic events regardless of their size. dnSNARE peptide, coding the cytoplasmic domain of Sb2, stabilized the fusion-pore diameter to narrow, release-unproductive diameters in both vesicle types, regardless of vesicle diameter. These experiments revealed two functional types of vesicles in astrocytes that contain distinct gliosignaling molecules. Given the slowness of regulated exocytosis in astrocytes and the targeting of slow cortical activity associated with dnSNARE expression in vivo, the behavioral phenotype resulting from inhibiting vesicular release is likely mainly associated with impaired release from larger vesicles containing ATP.

17:40 Session 1 Shuzo Sugita

UNC-18 and Tomosyn antagonistically act in parallel to control synaptic vesicle priming downstream of UNC-13.

Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examined how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in C. elegans, and increased synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, the unc-18(P334A) mutation partially bypasses the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations exhibit a strong synergy in suppressing unc-13 mutant defects. Biochemically, Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as increased binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts in parallel with Tomosyn, which inhibits exocytosis.

18:00 Poster and Exhibitor Presentations (1)
19:30 Welcome BBQ

Wednesday 23rd August

Session number
09:00 Plenary 2 Jakob Sorensen

On the involvement of synatotagmins and SNAREs in vesicle priming and fusion.

Regulated exocytosis involves several steps, including vesicle priming and fusion triggering. Priming is the process that makes the vesicle release-competent, and it was shown to be Ca2+-dependent in the sub-M range 25 years ago. Molecularly, it involves the assembly of the N-terminal end of the SNARE-complex between vesicle and plasma membrane with the help of Munc18 and Munc13 isoforms. Fusion triggering is also Ca2+-dependent (in the several-M range), and it requires Ca2+-binding to synaptotagmins (in chromaffin cells synaptotagmin-1 and synaptotagmin-7), and is assisted by the cytosolic protein complexin. I am going to discuss two questions. First: how is priming made Ca2+-dependent? Ca2+-dependent priming in chromaffin cells potentially allows circulating hormones that release Ca2+ from intracellular stores (e.g. histamine), to increase primed vesicle pool size, and act synergistically with neuronal stimulation to boost release . We have found that when present alone synaptotagmin-1 and synaptotagmin-7 both act autonomously in exocytosis triggering, with synaptotagmin-1 being the faster sensor. However, when present together, synaptotagmin-7 and synaptotagmin-1 take over different functions: synaptotagmin-7 now acts to decrease unpriming (the opposite reaction to priming), whereas synaptotagmin-1 acts to trigger fast release. I will discuss how these different functions might result from isoform competition in the presence of a conserved mechanism for synaptotagmin action. The second question is: what is the nature of the interaction between the SNAREs and synaptotagmins, which makes fusion Ca2+-dependent? This involves experiments performed in autaptic glutamatergic neurons. Using mutagenesis, we find that fusion triggering is limited by an electrostatic energy barrier, which is influenced by charges on the outside of the SNARE-bundle. This energy barrier for release is increased in amplitude by negative charges and decreased by positive charges. This makes it possible for Ca2+ to trigger release in a fraction of a millisecond by throwing an electrostatic switch.

Lipids and the regulation of Exocytosis
Chaired by Prof Nicolas Vitale
10:30 Session 2 Geert van den Bogaart

Quantitative visualization of SNARE complex formation in living cells.

Members of the SNARE protein family are essential for secretion and transport between organelles in all eukaryotic cells. However, for most of the intracellular trafficking routes, the SNARE proteins remain unidentified, because SNARE proteins are highly expressed and have multiple overlapping functions. This makes it complicated to identify the sets of SNARE proteins for specific intracellular membrane trafficking routes. Here, I will describe a novel technique based on Förster resonance energy transfer (FRET) fluorescence lifetime imaging microscopy (FLIM) allowing for quantitative visualization of SNARE complexes within living cells with organellar resolution. We used this FRET-FLIM technique to investigate which SNARE proteins are responsible for the secretion of the inflammatory cytokine interleukin-6 by dendritic cells. We found that upon bacterial encounter, dendritic cells have increased complexing of the SNARE proteins syntaxin 4 and VAMP3 specifically at the plasma membrane. Silencing of the gene for VAMP3 reduced the amount of secreted interleukin-6.

11:00 Session 2 Stephane Gasman

Regulation of transbilayer phospholipids scrambling controls exo-endocytosis coupling.


11:30 Session 2 Meyer Jackson

The protein-lipid transition in endocrine fusion pores – roles for synaptophysin.

Fusion pores provide a window into the mechanisms by which cells regulate secretion. Fusion pores start off as ion channel-like structures lined by protein transmembrane segments and then enlarge to form a meta-stable pore composed of lipid. Synaptophysin is a protein that resides on secretory vesicles and contains four transmembrane domains. To evaluate the role of synaptophysin in exocytosis and assess its involvement in fusion pores, amperometry recordings were made from mouse chromaffin cells from synaptophysin knockout mice, and from double knockout mice lacking synaptophysin and the related protein synaptogyrin. Recordings were also made from cells expressing wild type and mutant synaptophysin constructs. In the absence of both synaptophysin and synaptogyrin fusion events were less frequent, the initial fusion pore had a longer lifetime, and fusion pore openings often ended with closures to culminate in kiss-and-run. Synaptophysin also influenced the dynamics of later stages of exocytosis when fusion pores expanded to a presumably lipidic state. These results suggest that members of the synaptophysin family influence both the initial proteinaceous fusion pore as well as the downstream lipidic fusion pore. The C-terminal domain and transmembrane domain play different roles in different stages in the formation and expansion of fusion pores.

12:00 Session 2 Alexander Walter

Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis, the Ca2+-dependent fusion of intracellular vesicles with the plasma membrane. This process underlies chemical synaptic transmission and release of water-soluble hormones from endocrine cells. Classical ways of manipulating PI(4,5)P2 levels are slower than the rate of PI(4,5)P2 metabolism, making it hard to tease the roles of PI(4,5)P2 apart from those of its metabolites, including diacylglycerol (DAG), or other phosphatidylinositols. Here, we develop a membrane-permeant, photoactivatable PI(4,5)P2, which can be loaded into cells in an inactive form and rapidly activated by light, allowing sub-second specific increases in PI(4,5)P2 levels. By combining this compound with genetic manipulations and electrophysiological measurements in mouse adrenal chromaffin cells, we identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as essential PI(4,5)P2 effectors for exocytosis in live cells. Strikingly, uncaging of PI(4,5)P2 but not of DAG triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in secretion triggering. Thus, optical uncaging of signaling lipids can uncover their direct and rapid effects on cellular processes and identify lipid effectors.

12:30 LUNCH
14:00 Plenary 3 Margarent Robinson

Vesicle coats: what do they all do?

Vesicular traffic from one membrane compartment to another requires coat proteins, which shape the membrane into a vesicle and select the vesicle cargo. Since the discovery of the clathrin coat over 40 years ago, we have learned that there are at least half a dozen different types of coated vesicles, and over 100 different coat proteins. What is less clear, however, is what all the coats are actually doing. Approaches that can now be used to address this question include siRNA knockdowns, knockouts via genome editing, and knock sideways to remove the available pool of a protein within minutes. Readouts for the resulting phenotypes include proteomic analyses of both vesicles and the entire cell, making it possible to look for changes in the subcellular distribution of thousands of proteins. In this way, we are gradually building up a picture of coat protein function in different cell types, including chromaffin cells. We are also beginning to understand what goes wrong when a coat protein is missing in a genetic disorder, or is hijacked by a pathogen.  

Secretory Vesicles - from the inside out
15:00 Session 3a Ricardo Borges

Inside a chromaffin granule

The occurrence of large concentrations of neurotransmitters inside chromaffin granules, which by far exceed the theoretical tonicity of cytosol has captivated the attention of scientists along decades. For instance, chromaffin granules are able to accumulate, along with many other products, near molar concentration of catecholamines, hundreds of millimolar of ATP and notable concentrations of calcium, ascorbate, peptides, nucleotides and chromogranins thus forming a vesicular cocktail. We will expose our current view of the interactions of vesicular components emphasizing in their role in the accumulation and exocytosis of catecholamines. We will present compiled data shown the direct implication of the soluble species concentrated in the granule content in the kinetics of exocytosis. We will focus our attention on chromogranins (by altering endogenous expression), ATP (by acting on the expression of the vesicular nucleotide carrier) and pH (by pharmacological approach) in these crucial physiological functions.

15:30 Session 3a Yongsoo Park

MicroRNA exocytosis by vesicle fusion in chromaffin cells

Neurotransmitters and peptide hormones are secreted into outside the cell by a vesicle fusion process. Although non-coding RNA (ncRNA) that include microRNA (miRNA) regulates gene expression inside the cell where they are transcribed, extracellular miRNA has been recently discovered outside the cells, proposing that miRNA might be released to participate in celltocell communication. Despite its importance of extracellular miRNA, the molecular mechanisms by which miRNA can be stored in vesicles and released by vesicle fusion remain enigmatic. Using next-generation sequencing, vesicle purification techniques, and synthetic neurotransmission, we observe that large dense-core vesicles (LDCVs) contain a variety of miRNAs including miR-375. Furthermore, miRNA exocytosis is mediated by the SNARE complex and accelerated by Ca2+. Our results suggest that miRNA can be a novel neuromodulator that can be stored in vesicles and released by vesicle fusion together with classical neurotransmitters.

Chromogranins in cancer and metabolism
Chaired by Prof Angelo Corti & Sushil Mahata
16:30 Session 3b Angelo Corti

Role of Neuropilin-1 in Chromogranin A-mediated regulation of Angiogenesis and tumor growth

We have previously shown that full-length chromogranin A (CgA1-439), a secretory protein released in circulation by the neuroendocrine system, can inhibit angiogenesis, whereas the truncated form CgA1-373 is pro-angiogenic. CgA1-439 and CgA1-373 are released in variable amounts in the blood of cancer patients. We investigated the receptor mechanisms underlying the CgA1-373 pro-angiogenic activity. In vitro biochemical studies showed that CgA1-373 can bind to neuropilin-1. Structure function studies showed that the neuropilin-1 binding site is located in the truncated C-terminal region of the CgA1-373 (CgA368-373: GPGPQLR) and that R373 is crucial for the binding. In vitro angiogenesis assays showed that the pro-angiogenic activity of CgA1-373 could be inhibited by antibodies against neuropilin-1, and by antibodies against the GPGPQLR truncated epitope of CgA1-373, pointing to a crucial role of this receptor. The pro-angiogenic activity of CgA1-373 was also inhibited by anti-VEGF and anti-FGF-2 antibodies. In vivo studies showed that antibodies against the GPGPQLR site of CgA1-373, which is formed during tumor progression, can reduce tumor perfusion and growth, suggesting that CgA fragmentation and interaction with neuropilin-1 is crucial for tumor angiogenesis and progression. Finally, we found the C-terminal residue R373, crucial for binding, is rapidly cleaved to generate CgA1-372, with consequent loss of neuropilin-1 binding and gain of potent anti-angiogenic activity. Overall, these results suggest that cleavage of CgA at the R373R374 dibasic site in tumors and the subsequent removal of R373 in plasma represents an important “off/on/off” switch for the regulation of angiogenesis in tumors, thereby representing a novel therapeutic target.

16:50 Session 3b Sushil K. Mahata

Catestatin Inhibits obesity-induced macrophage infiltration and inflammation in the liver and suppresses hepatic glucose production leading to improved insulin sensitivity

Macrophages (MΦs), one of the cells of the innate immune system, reside in or infiltrate metabolic organs under obese conditions and contribute to low-grade inflammation that impairs insulin action, leading to the development of insulin resistance. MΦs play crucial roles in hepatic inflammation and insulin resistance as supported by improvement in insulin sensitivity after chemical deletion or genetic impairment of hepatic MΦs. Here, we have investigated the role of chromogranin A (CgA) peptide catestatin (CST) in regulation of MΦ function and the consequent improvement in insulin action. The activation of resident Kupffer cells (KCs) and monocyte (Mc)-derived recruited macrophages (McMΦs) in the liver contributes to obesity-induced insulin resistance and type 2 diabetes. FACS-sorted F4/80+Ly6C- cells (representing KCs) and F4/80-Ly6C+ cells (representing McMΦs) revealed increased nuclear/cytoplasmic ratio in McMΦs compared to KCs. Diet-induced obese (DIO) mice treated with catestatin (CST) inhibited the infiltration of McMΦs, decreased expression of pro-inflammatory genes, and increased expression of anti-inflammatory genes in MΦs, resulting in improvement in glucose and insulin tolerance in DIO mice. Systemic CST knockout (CST-KO) mice on normal chow diet (NCD) ate more food, gained weight, and displayed elevated blood glucose and insulin levels. Supplementation of CST normalized glucose and insulin levels. In order to verify that CST deficiency caused macrophages to be very pro-inflammatory in CST-KO mice (even being under NCD) and produced glucose intolerance, we tested the effects of MΦs on hepatic glucose production (HGP). Both basal and glucagon-induced HGP was markedly increased in hepatocytes co-cultured with KCs and McMΦs from NCD-fed CST-KO mice, and the effect was abrogated upon pre-treatment of CST-KO-MΦs with CST. The above findings provide a novel mechanism of HGP suppression through CST-mediated inhibition of macrophage infiltration and function and the consequent improvement in peripheral insulin sensitivity.

17:00 Session 3b Youssef Anouar

New functions for granins in the neuroendocrine system

Granins are a family of secretory glycoproteins present in endocrine and neuroendocrine cells whose members chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII) among others have been the subject of intense investigations for their roles as prohormones and as important players of granulogenesis, but whose precise functions are still a matter of debate. Large evidence has been gained showing that these major constituents of secretory vesicles play a role in the formation of large dense core secretory granules at the level of the Golgi apparatus. We have recently shown that CgA interacts with lipids and components of the cytoskeleton including myosin 1B to trigger the budding of secretory granules in the trans Golgi network (TGN). Indeed, CgA displays a high affinity for specific phosphatidic acid entities, whose inhibition impairs secretory granule biogenesis and hormone release from neuroendocrine cells and CgA-induced secretory granules in non-endocrine cells. Likewise, our proteomic and functional studies revealed that myosin 1B and F actin are also involved in the control of CgA-induced secretory granule budding from the TGN, further supporting the notion that granins in association with different actors play a central role in neurosecretion. In addition, granins give rise to several regulatory peptides which exert their activities in the brain and periphery. We have recently shown that a SgII-derived peptide that we have characterized in different tissues and species and named EM66, exerts an anorexigenic effect after intracerebroventricular administration. This effect is associated with an increase in POMC and MC3R mRNA levels, which constitutes the anorexigenic system, and c-Fos immunoreactivity in the arcuate nucleus of the hypothalamus. In addition, SgII as well as POMC mRNA levels are down-regulated in the hypothalamis after feeding mice a high-fat diet, further supporting a role of EM66 in the control of food intake. Together, the present data further support the view that granins are multifunctional proteins able to control cell secretion through their involvement in the initial steps of granulogenesis and to exert neuroendocrine activities via several regulatory peptides with an impact in pathophysiology.

17:30 Break
17:45 Poster Presentations (2)
19:00 Wine & Cheese Events
Chaired by Ricardo Borges

Thursday 24th August

Session number
Morning Excursions
SNARE structure and function
Chaired by Prof Sebastian Bara
14:00 Session 4a Manfred Lindau

The exocytotic fusion pore formed by the neuronal SNARE complex

The neuronal SNARE complex, which in mammalian neurosecretory cells is composed of the vSNARE synaptobrevin 2 (Syb2), and the tSNAREs syntaxin-1 and SNAP25, plays a key role in vesicle fusion. However, in spite of an enormous body of research the molecular mechanism of fusion pore formation and the structure of the fusion pore are still not clear. In individual chromaffin cells, we tracked conformational changes in SNAP25 by total internal reflection FRET microscopy while exocytotic catecholamine release from single vesicles was simultaneously recorded using a microfabricated electrochemical detector array. A local rapid and transient FRET change lasting ~5 s occurred precisely where individual vesicles released catecholamine. The FRET change was abrupt and preceded the opening of an exocytotic fusion pore by ~60-90 ms. A similar transient FRET change can be stimulated by pulse depolarization, coincides with the [Ca2+] change and requires the v-SNARE. The results indicate that the Ca2+ signal induces a rapid conformational change in the SNARE complex, followed by fusion with a delay that may depend on [Ca2+]. Experiments with Syb2 transmembrane domain mutations and molecular dynamics (MD) simulations support the hypothesis that in neurosecretory cells, fusion pore formation is directly accomplished by a conformational change in the SNARE complex via movement of the transmembrane domains. Coarse Grained MD simulations of SNARE protein mediated fusion pore formation between a nanodisc and a planar membrane, revealed formation of proteolipid fusion pores. Estimates of the conductances of these fusion pores are in excellent agreement with experimentally measured early fusion pore conductances. Combining ion substitution and amperometry as well as MD simulations indicate that fusion pores <~200 pS are cation selective and become non-selective as the fusion pore expands. The results suggesting that the fusion pore structures evolving in these simulations reflect the molecular structure of exocytotic fusion pores.

14:15 Session 4a Yongil Zhang

Regulated SNARE folding/assembly in synaptic exocytosis.
Sponsored by the Biochemical Society

All actions and thinking depend on delicate machinery at synapses that release neurotransmitters with extremely high speed and precision. Core components of the release machinery include SNARE proteins, Munc18-1, and synaptotagmin (Syt). SNAREs couple their stage-wise folding and assembly to synaptic vesicle fusion like a zipper, and Munc18-1 and Syt regulate the SNARE assembly. How these components work together to achieve the high speed and precision required for neurotransmission remains unclear. Using high-resolution optical tweezers, we demonstrate that SNAREs zipper in at least two distinct stages-slow N-terminal domain association and fast C-terminal domain zippering. Assembly of a single SNARE complex generates total energy of 70 kT, sufficient to overcome the energy barrier for membrane fusion. Results of layer mutations suggest that the N-terminal and the C-terminal domains are responsible for vesicle docking and fusion, respectively. Munc18-1 enhances SNARE assembly by initiating SNARE pairing like a matchmaker. We also developed a single-molecule assay to characterize the calcium- and lipid-dependent binding between synaptotagmin and membranes. Our study revealed that synaptotagmin facilitates membrane fusion by regulating the distance between apposed membranes. Thus, we corroborate step-wise SNARE assembly and their distinct functions in synaptic vesicle fusion and clarify the regulatory roles of Munc18-1 and synaptotagmin in SNARE assembly and neurotransmitter release.

14:45 Session 4a Sebastian Barg

Regulation of insulin granule fusion pore expansion

Exocytosis proceeds through an intermediate state in which a narrow fusion pore connects the secretory vesicle lumen with the extracellular environment. Small molecules such as nucleotides can escape through the fusion pore, while larger peptide hormones such as insulin remain trapped and require pore expansion for release. There is evidence that pore restriction/expansion is a regulated process involving Ca2+ and the GTPase dynamin. We have studied fusion pore expansion in insulin secreting beta-cells using high resolution imaging, by exploiting the luminal pH switch that occurs just after pore opening. I will present recent data detailing the mechanism by which cAMP dependent hormone action affects pore expansion, as well as a results from a systematic screen of proteins involved in exo- and endocytosis where we asked whether expression of certain proteins affect fusion pore behavior and whether they are present at the site of restricted pores. The data have implications for the understanding of two major antidiabetic drugs and certain genetic alleles that increase the risk for development of type-2 diabetes.

15:00 Session 4a Nikhil Gandasi

Disturbed association of Ca2+ channels with insulin-containing granules during type 2 diabetes

Loss of first-phase insulin secretion is an early sign of developing type 2 diabetes (T2D). Ca2+ entry through voltage-gated L-type Ca2+ channels triggers exocytosis of insulin-containing granules in pancreatic β cells and is required for the postprandial spike in insulin secretion. Using high-resolution microscopy, we have identified a subset of docked insulin granules in human β cells and rat-derived clonal insulin 1 (INS1) cells for which localized Ca2+ influx triggers exocytosis with high probability and minimal latency. This immediately releasable pool (IRP) of granules, identified both structurally and functionally, was absent in β cells from human T2D donors and in INS1 cells cultured in fatty acids that mimic the diabetic state. Upon arrival at the plasma membrane, IRP granules slowly associated with 15 to 20 L-type channels. We determined that recruitment depended on a direct interaction with the synaptic protein Munc13, because expression of the II–III loop of the channel, the C2 domain of Munc13-1, or of Munc13-1 with a mutated C2 domain all disrupted L-type channel clustering at granules and ablated fast exocytosis. Thus, rapid insulin secretion requires Munc13-mediated recruitment of L-type Ca2+ channels in close proximity to insulin granules. Loss of this organization underlies disturbed insulin secretion kinetics in T2D.

15:15 Session 4a Arun Anantharam

Secretory granule heterogeneity and the regulation of calcium-triggered exocytosis.

Adrenomedullary chromaffin cells respond to sympathetic nervous system activation by secreting a cocktail of potent neuropeptides and hormones into the circulation. The distinct phases of the chromaffin cell secretory response have been attributed to the progressive fusion of distinct populations of dense core granules with different activation kinetics. However, it has been difficult to define what distinguishes these populations at the molecular level. Functional segregation of granule pools may depend on selective sorting of synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7), which our previous work showed are rarely cosorted to the same granule. Here we assess the consequences of selective sorting of Syt isoforms in chromaffin cells, particularly with respect to granule dynamics and activation kinetics. Upon depolarization of cells expressing fluorescent Syt isoforms using elevated K+, we find that Syt-7 granules fuse with faster kinetics than Syt-1 granules, irrespective of stimulation strength. Pharmacological blockade of Ca2+ channels reveals differential dependence of Syt-1 versus Syt-7 granule exocytosis on Ca2+ channel subtypes. Syt-7 granules also show a greater tendency to fuse in clusters than Syt-1 granules, and granules harboring Syt-1 travel a greater distance before fusion than those with Syt-7, suggesting that there is spatial and fusion-site heterogeneity among the two granule populations. However, the greatest functional difference between granule populations is their responsiveness to Ca2+. Upon introduction of Ca2+ into permeabilized cells, Syt-7 granules fuse with fast kinetics and high efficacy, even at low Ca2+ levels (e.g., when cells are weakly stimulated). Conversely, Syt-1 granules require a comparatively larger increase in intracellular Ca2+ for activation. At Ca2+ concentrations above 30 μM, activation kinetics are faster for Syt-1 granules than for Syt-7 granules. Our study provides evidence for functional specialization of chromaffin cell granules via selective expression of Syt isoforms with different Ca2+ sensitivities.

15:30 Session 4a Cordelia Imig

Molecular Mechanisms of LDCV Docking and Priming.

Secretory vesicle docking, priming, and Ca2+-triggered fusion are orchestrated by a complex and highly conserved molecular machinery. Despite the similarities in the composition of the release machinery between different neurosecretory systems, their functional and morphological characteristics can differ dramatically, for example, with respect to the ultrastructural architecture of release sites and transmitter release properties. The accurate assessment of vesicle docking requires electron microscopy to resolve inter-membrane distances in the nm range, but information on proteins involved in this process has been partly inconclusive. Important reasons for experimental inconsistencies are that diverse preparations, cell types, sample fixation methods, imaging approaches, and docking definitions have been employed. Combining organotypic slice cultures from mice lacking key proteins of the release machinery, rapid cryofixation, freeze substitution, and 3D-electron tomography of hippocampal spine synapses, we found that synaptic vesicle (SV) docking requires Munc13 proteins and all three neuronal SNAREs (SNAP-25, Synaptobrevin-2, Syntaxin-1), but not necessarily Synaptotagmin (Syt)-1. Our data indicate that morphologically docked SVs comprise the readily-releasable pool (RRP) of primed SVs and loss of Munc13 and SNARE proteins cause an accumulation of SVs in close-distance of the membrane that are possibly “tethered” to the active zone. However, loss of Munc13 proteins in chromaffin cells had no effect on large dense-core vesicle (LDCV) docking despite a severe reduction in the functional RRP. Similarly, loss of the neuronal SNAREs SNAP-25 and Synaptobrevin-2 did not alter LDCV docking, indicating that the majority of docked LDCVs in chromaffin cells are not fusion-competent and that we may not be able to morphologically distinguish the functional RRP in this neurosecretory system. Interestingly, we further dissected two distinct pools of “docked” and “tethered” LDCVs within 40 nm of the membrane in all genotypes analysed, highlighting the need for high-resolution 3D-electron microscopy approaches to accurately assess LDCV docking in chromaffin cells.

Mechanisms of Endocytosis
Chaired by Prof Manfred
16:00 Session 4b Shigeki Watanabe

Ultrafast endocytosis of synaptic vesicles

In the early 70s, two models for synaptic vesicle endocytosis were put forward based on the ultrastructural studies of frog neuromuscular junctions. Heuser and Reese found that vesicles fuse and collapse into the membrane and synaptic vesicles are recovered via slow, clathrin-mediated endodcytosis. Ceccarelli and his colleagues observed a fusing vesicle with a narrow neck and deduced that vesicles do not fully collapse into the membrane and that vesicles are recovered thorough a rapid reversal of the fusion pore. Since then, conflicting evidence has accumulated: the molecular studies suggest that clathrin and clathrin-associated proteins are essential, but the kinetics studies have indicated the existence of both fast and slow mechanisms. The major criticism of the original morphological studies was that they used intense stimulation to ensure the capture of endocytic events. To determine how endocytosis takes place after a single action potential, we coupled channelrhodopsin-induced synaptic transmission with rapid high-pressure freezing and captured membrane dynamics following fusion of synaptic vesicles. We found that vesicle membrane is retrieved within 100 ms after fusion in mouse hippocampal neurons. This ultrafast endocytic pathway is compensatory – the amount of membrane retrieved equals the amount of membrane exocytosed. Following ultrafast endocytosis, large endocytic vesicles are delivered to endosomes. Synaptic vesicles are then regenerated from the endosomes ~5 s after stimulation. The newly formed vesicles return to active zone, suggesting that these vesicles are functional synaptic vesicles. To test the requirement for clathrin, we generated shRNA against clathrin heavy chain. We found that ultrafast endocytosis is intact in the clathrin knock-down cells. However, synaptic endosomes were not resolved into synaptic vesicles, suggesting that clathrin is required for regenerating vesicles from endosomes. These results suggest that regeneration of synaptic vesicles is a two-step process: rapid, clathrin-independent internalization of vesicle membrane followed by slower, clathrin-dependent reconstitution of vesicles.

16:30 Session 4b Jenny Hinshaw

Capturing the sequential steps of dynamin-mediated fission by cryo-EM.

Our laboratory is interested in membrane fusion and fission mediated by dynamin and other dynamin-like proteins. We are currently focusing on dynamin and its role in endocytosis. The approach we are taking is to examine the structure and function of proteins involved in these processes by cryo-electron microscopy (cryo-EM), and biochemical methods. In addition to examining the gross morphology of protein complexes, we have solved the 3-dimensional structure of several dynamin family members by image processing methods. We currently have a 4.3 Å resolution cryo-EM structure of the constricted, membrane-associated helical polymer of human dynamin-1 in the GTP bound state. Compared to the soluble tetramer, the membrane-associated dynamin adopts a more extended conformation. In addition, our new model fits well into the cryo-EM map of a constricted, membrane-free spiral, suggesting membrane is dispensable for stabilizing the constricted conformation. The model also fits well into the map of a super-constricted polymer, confirming that the new interfaces are sufficient to bring the lipid bilayer close enough for spontaneous fission. The new reconstruction also allows unambiguous placement of dynamin’s membrane-binding pleckstrin homology domain, which has been poorly defined in previous models due to flexibility. Overall, our new structure, in combination with previous 3D reconstructions in different nucleotide states, capture conformational changes in the polymer that ultimately leads to membrane fission.

17:00 BREAK
Early Career Researchers' Symposium
Molecular Organization of exocytosis observed with super-resolution imaging
Chaired by Profs Frederic Meunier & Nicolas Vitale
17:15 Adekunle Bademosi

In vivo single molecule imaging of Syntaxin1A in motor nerve terminals

Syntaxin1A is a key protein involved in mediating synaptic transmission through its ability to form the SNARE complex with cognate partners – SNAP-25 and VAMP2. Syntaxin1A molecules on the plasma membrane of neuro-secretory cells are organized in nano-clusters, which have been shown to play an important role in docking and priming of secretory vesicles. How individual molecules of Syntaxin1A enter and exit these nano-clusters by lateral diffusion and how stimulation affects their dynamic equilibrium at the pre-synapse in vivo in unknown. To image single molecules of Syntaxin1A in live synapses, we generated a Drosophila line constitutively expressing photo-convertible fluorescently tagged Syntaxin1A (Sx1A-mEos2) to carry out single particle tracking Photoactivated Localization Microscopy on live Drosophila larva neuromuscular junction. We investigated the change in Syntaxin1A mobility and nano-domain organization elicited by increased pre-synaptic activity using both opto-genetic and thermo-genetic tools. Here, we show that in Drosophila larva motor nerve terminals, the overall mobility of Syntaxin1A molecules was low and raising synaptic activity led to an increase in Syntaxin1A mobility. This suggested that a significant proportion of Syntaxin1A molecules are pre-engaged in the SNARE complex, which confer such relatively low mobility. Indeed, concomitant expression of tetanus toxin light chain, which prevents SNARE complex assembly, significantly increased Syntaxin1A mobility, but opposing effect on Syntaxin1A mobility was observed when SNARE complex disassembly was prevented by the temperature sensitive NSF mutation. Decreasing Syntaxin1A interaction with polyphosphoinositides abrogated the activity-dependent increase in mobility. Our results suggest that the relative immobility of Syntaxin1A molecules within synapses is indicative of a high level of primed vesicles in live motor nerve terminals.

17:30 Sebastien Houy

Doc2B acts as a calcium sensor for vesicle priming requiring synaptotagmin-1, Munc13-2 and SNAREs

Doc2B is a cytosolic protein with two C2-domains that bind to membranes, Ca2+ and SNAREs, and with further binding sites for Munc13 and the dynein light-chain Tctex-1. The function of Doc2B as a calcium sensor akin to synaptotagmins, and its involvement in different phases of release, is under debate. Here, we employed mutational analysis of candidate Doc2b interaction partners, combined with membrane capacitance measurements and carbon-fiber amperometry to dissect the role of Doc2B in neuroendocrine cells. In mouse adrenal chromaffin cells both the fast and slow bursts of release were reduced in the absence of Doc2B, whereas release during sustained calcium elevations was potentiated. Cross-rescue experiments showed that Doc2B does not substitute for the two main Ca2+ sensors for release: synaptotagmin-1 and -7. Substitution of the Ca2+-coordinating aspartates with asparagine (C2A-domain DN mutation) or alanine (C2A/B-domain 6A mutation) blocked Ca2+-dependent Doc2B translocation, and rendered vesicle priming Ca2+-independent. Mutations designed to interfere with SNARE (KE mutant), or Munc13 (mutation: N-terminal part of the MID-domain) interaction blocked the stimulation of priming, and so did the elimination of ubMunc13-2 or synaptotagmin-1. Mutation of the Tctex-binding site had only subtle effects. None of those mutations affected the function of Doc2B in inhibiting sustained release. However, deleting the entire MID domain (MID, aa 14-41) relieved the inhibitory effect, but inhibition persisted in the absence of ubMunc13-2. Overall, we conclude that Doc2B acts as a calcium-dependent priming factor, and that the inhibitory and stimulatory effects of Doc2B involve overlapping sequences near the MID-domain, pointing to a complex interaction with the release machinery.

17:45 Merja Joensuu

Imaging live synaptic vesicles by subdiffractional tracking of internalized molecules (sdTIM).

While the resolution of conventional optical microscopy is diffraction-limited, isolated light-emitting molecules can be localized within a far smaller volume using nanoscopic methods. Advances in imaging methodologies have provided crucial information on neuronal endocytic pathways, increasing our understanding of synaptic vesicle (SV) recycling, synaptic activity, neuronal survival and homeostasis. One of the main limitations of the current super-resolution technologies for direct visualization of SVs in presynapses is their limited ability to track multiple vesicles simultaneously which dramatically restrict the dissection of SV discrete diffusional and transport states. To address this, we implemented a novel pulse-chase technique based on the subdiffractional tracking of internalized molecules (sdTIM). The technique allowed us to image anti-green fluorescent protein Atto647N-tagged nanobodies trapped in SVs from live hippocampal nerve terminals expressing vesicle-associated membrane protein 2 (VAMP2)–pHluorin (Joensuu et al., JCB 2016). We have further extended our protocol for sequential dual-colour super-resolution imaging of Atto565-tagged nanobodies and Alexa647-tagged cholera toxin subunit-B internalized in SVs and signalling endosomes, respectively, undergoing long-range axonal retrograde transport with 30-50 nm localization precision (Joensuu et al., Nat. Protocols in press). Our results showed that, once internalized, VAMP2-pHluorin/Atto647N-tagged nanobodies exhibited a markedly lower mobility than on the plasma membrane, an effect that was reversed upon restimulation in presynapses but not in neighbouring axons. Combining sdTIM with Bayesian model selection applied to hidden Markov modelling, allows determining both (i) the number of active transport and diffusive states underlying a particular particle trajectory, and (ii) when transitions between these states occur. Our results showed that SVs oscillate between diffusive states, or a combination of diffusive and transport states with opposite directionality. Importantly, SVs exhibiting diffusive motion were less likely to switch to the transport motion. sdTIM technique offers a transferable approach to track additional subdiffractional endocytic structures in live neurons and other cell types.

18:00 Sumana Mahata

Chromogranin A and its peptide catestatin regulates catecholamine storage and catecholamine granule morphology.

Chromogranin A (CgA) is co-stored and co-released with catecholamines from dense core (DC) vesicles (DCV) in the adrenal medulla and regulates biogenesis of DCV. CgA peptide catestatin (CST), however, inhibits nicotine-induced catecholamine secretion from DCV. Here, we report that CgA and CST regulate catecholamine storage and DCV morphology in mouse adrenal medulla and rat PC12 cells, respectively.

CgA knockout (Chga-KO) mice contain 30-40% less catecholamines in their adrenal medulla compared to wild-type (WT) mice. Concomitant with decreased catecholamines, the DCV diameter in Chga-KO mice was less (100-200 nm) than in WT mice (200-350 nm). Chga-KO mice also showed an ~47% increase in DCV/DC area ratio, implying vesicle swelling due to increased osmotically active free catecholamines. In PC12 cells, the average diameter of DCV and DC were 142.07±1.31 and 105.77±1.07 nm, respectively. The preponderant DCV and DC diameters were 100-150 nm and 50-100 nm, respectively. Treatment of PC12 cells with peptidergic transmitter PACAP (0.1 µM) for 24 hours resulted in ~47% decrease in 100-150 nm DC diameter. Co-treatment of CST and PACAP caused ~20% increase in 100-150 nm DC diameter. Stimulation of PC12 cells by nicotinic-cholinergic agonist nicotine (60 µM) resulted in ~47% decrease in 50-100 nm DC diameter. Co-treatment of CST and nicotine caused ~23% increase in 50-100 nm DC diameter. CST decreased DCV/DC area ratio by ~13% in PACAP-treated and ~31% in nicotine-treated PC12 cells.

We conclude that CgA and CST regulate catecholamine storage and catecholamine storage granule morphology in mice and PC12 cells.

#The Noland Scholarship from Caltech supported Sumana Mahata.

18:15-18:30 Rachel Gormal

Fluorescent intra-body localization Microscopy (FiLM): A novel method for tracking single intracellular endogenous and GFP-tagged proteins in vitro and in vivo

By breaking the Abbe law of diffraction, super-resolution microscopy techniques provide unprecedented detail of biological structures and processes. However, the requirement for fluorescent photoconvertible tags has hampered progress and exposed PALM and sptPALM techniques to over-expression artifacts, raising the need for developing novel tools to bypass these limitations. Herein, we describe the development of single chain expressed in cells as ‘intra-nanobodies’ to perform single molecule imaging of any GFP-tagged or endogenous intracellular proteins. Configuration 1: Co-expression of a GFP binding nanobody tagged with a photoconvertible mEOS2 to image any GFP-tagged protein in cells. We co-expressed anti GFP-intra-nanobody-mEos with PH-PLCδ-GFP allowed super-resolution imaging of phosphatidylinositol(4,5)bisphosphate nanodomains in fixed and live neurosecretory PC12 cells. We found identical domain size and mobility when using PH-PLCδ-mEos2. Further, combining the intra-nanobody with an Apex tag allowed us to perform 3D electron microscopy on these nanodomains. Using the same technique, we visualized cell-cell junctions at nanoscale in genetically modified CACO2 cell line expressing GFP tagged E-cadherin at endogenous levels. Expressing the GFP intra-nanobody, within the nematode C. elegans PLM mechanosensory neurons, allowed us to visualize the fusogen protein EFF-1 at nanoscale in vivo. In addition, using zebrafish D. rerio expressing the GFP intra-nanobody, we were able to visualize Cavin 1 within its muscle fibers. Configuration 2: Purpose-designed intra-nanobodies to probe the nanoscale organization of endogenous proteins. Two specific nanobodies developed against the endogenous β2 adrenoreceptor, Nb80 and Nb37, were used to track endogenous receptors either in their active or inactive states.

Fluorescent intra-body Localization Microscopy (FiLM), therefore enables GFP-tagged constructs to be localized by super-resolution microscopy, avoiding cloning and reagent requirements for existing in vitro or in vivo models. This technique can also be extended to identify the diffusional signature and nanoscale organization of endogenous proteins by expressing selective purpose-designed intra-nanobodies.

18:45 ISCCB International Organizing Committee Meeting

Friday 25th August

Session number
09:00 Plenary 4 Stephen Hill

Investigating ligand-receptor interactions at GPCRs in living cells using fluorescent ligands

G protein-coupled receptors control a wide range of physiological processes and are the target for many clinically used drugs. Understanding the way in which receptors bind agonists and antagonists, their organisation in the membrane and their regulation after agonist binding are important properties which are key to developing new drugs. One way to achieve this knowledge is through the use of fluorescent ligands, which have been used to study the expression and function of receptors in endogenously expressing systems. Fluorescent ligands with appropriate imaging properties can be used in conjunction with confocal microscopy to investigate the regulation of receptors after activation. Alternatively, through the use of single molecule microscopy, they can probe the spatial organisation of receptors within the membrane. In this talk I will present techniques in which fluorescent ligands have been used and the novel aspects of G protein-coupled receptor pharmacology which have been uncovered.

Chromogranins in vesicular and cardiac functions
Chaired by Profs Nitish Mahapatra & Sushill Mahata
10:15 Session 5 Annett Hellebo Ottesen

Functional aspects of catestatin and secretoneurin in cardiac disease

Cardiovascular disease (CVD) is a leading cause of morbidity and mortality throughout the world. Accumulating evidence suggests that the granin protein family plays an important role in cardiac pathophysiology and may serve as CV biomarkers. Secretogranin II (SgII) and chromogranin A (CgA) are cleaved by proteases to the short functional fragments; secretoneurin (SN) and catestatin (CST), respectively. We have combined clinical studies and experimental studies in cardiac cells and tissue to explore SN and CgA as disease markers and functional peptides in CVD. Circulating SN levels were found to be closely associated with mortality in patients with acute heart failure (HF) and ventricular arrhythmia-induced cardiac arrest. Furthermore, SN levels were increased in patients with CPVT. SN is internalized by endocytosis. Intracellularly, SN is a CaMKIIδ inhibitor that improves Ca2+-handling and attenuates Ca2+-dependent arrhythmogenesis. We provide data suggesting that high SN levels may be a compensatory protective mechanism in CVD via reduced CaMKIIδ activity. Myocardial CgA to CST conversion is impaired due to hyperglycosylation in HF and low CgA-to-CST conversion is associated with increased mortality in patients with acute HF. CST improves Ca2+ handling via CaMKIIδ inhibition. Thus, CgA hyperglycosylation in the failing myocardium may lead to adverse outcome due to unrestricted local CaMKIIδ control.

Accordingly, we have identified SN and CgA as CV biomarkers that provide additional prognostic information to established risk indices in patients with myocardial dysfunction and we have identified SN and CST as intracellular small peptide blockers of CaMKIIδ activity. CaMKIIδ is one of the most interesting future targets in HF, and SN and CST could prove to be interesting candidates for future CaMKIIδ inhibition.

10:30 Session 5 Sushil K. Mahata

Catestatin plays crucial roles in ischemic preconditioning-induced cardioprotection against ischemia/reperfusion injury.

Acute myocardial infarction (AMI), the major manifestation of coronary heart disease (CHD), is the leading cause of death and disability world-wide. The heart’s intrinsic ability to “condition” itself (endogenous cardioprotection) has emerged as a powerful strategy to limit myocardial injury, preserve left ventricular systolic function and potentially improve morbidity and mortality in patients with CHD. Currently, ischemic pre- and post-conditioning phenomena offer the strongest cardioprotection. Administration of catestatin (CST) at the onset of reperfusion and postconditioning has been shown provide comparable cardioprotection. To gain better insights into the role of CST in cardioprotection, we have generated systemic CST knockout (CST-KO) mice and evaluated cardioprotection induced by ischemic preconditioning (IPC).

Like chromogranin A knockout mice, CST-KO mice were also hypertensive and hyperadrenergic, indicating that the important role played by CgA in cardiovascular diseases relies at least in part on CST. In Langendorff-perfused heart preparation, IPC caused significant increase in left ventricular developed pressure (LVDP) in wild-type (WT) hearts compared to CST-KO hearts. Furthermore, IPC-induced CST-KO hearts did not show significant increase in LVDP. Consistent with LVDP, IPC also decreased LV end diastolic pressure (LVEDP) in WT hearts but not in CST-KO hearts. In addition, IPC-induced CST-KO hearts did not show any significant change in LVEDP, indicating the crucial roles played by CST in cardioprotection. CST-KO hearts show decreased glucose uptake and metabolism but increased fatty acid uptake and metabolism. This metabolic switch explains at least in part for the inability of CST-KO hearts for IPC-induced cardioprotection. Furthermore, CST-KO hearts exhibit decreased sarcomere length and attenuated expression of sarcomeric contractile genes Myl4, Myl7, and Myh7, and supplementation with CST restored the above abnormalities, implicating that CST is one of the key players in cardioprotection.

We conclude that CST is an indispensable endogenous peptide for cardioprotection.

10:45 Session 5 Nitish Mahapatra

Human chromogranin A gene promoter haplotypes: Differential interactions with the transcription factor c-Rel and implications for cardiometabolic disorders

Chromogranin A (CHGA), an acidic glycoprotein co-stored/co- secreted with catecholamines, plays a major role in the biogenesis of dense-core secretory vesicles in diffused neuroendocrine system. CHGA levels are elevated in several cardiovascular disease states but the underlying mechanisms are not well established. We aimed to identify common genetic variants in the promoter region of CHGA and to explore the mechanistic basis behind the plausible contribution of those variants in regulating CHGA protein levels in circulation. We re-sequenced the CHGA locus in 769 human subjects and discovered nine single nucleotide polymorphisms (SNPs) in the promoter region: G-1106A, A-1018T, T-1014C, T-988G, G-513A, G-462A, T-415C, C-89A and C-57T. In addition to the linkage disequilibrium (LD) observed among SNPs at -1014, -988, - 462 and -89 bp positions, we observed a strong LD between the A-1018T and C-57T SNPs. Haplotype analysis across CHGA promoter discovered five major promoter haplotypes. These haplotypes displayed differential promoter activities; specifically, haplotype 2 (containing variant T alleles at -1018 and -57 bp positions) showed the highest promoter activity. Computational and experimental analyses revealed the role of the transcription factor c-Rel in activating the CHGA promoter haplotype 2 under basal and pathophysiological conditions. In corroboration with the higher CHGA promoter activity of haplotype 2 in vitro, individuals carrying this haplotype showed higher plasma CHGA level; haplotype 2 subjects also displayed elevated body mass index (by ~2.4 Kg/m 2 , p=0.004), diastolic blood pressure (by ~6.0 mm Hg, p=0.026) and plasma glucose (by ~8.0 mg/dl, p=0.001) levels as compared to the most common haplotype (haplotype 1). Taken together, this study points towards a functional role of a common CHGA promoter haplotype (estimated to occur in ~900 million people Worldwide) and provides a molecular basis for enhanced expression of CHGA in haplotype 2 carriers who may be at higher risk for cardiovascular and metabolic disorders.

11:00 Session 5 Teresa Pasqua

Chromogranin A and its derived peptides: cardiac modulation and cardioprotection

The discovery of a cardiac production of Chromogranin A (CgA) in 1990 opened a fruitful field of research whose outcome is a cardiovascular dimension for this multifunctional protein, with great relevance in cardiac physio-pathology and clinics. In the rat heart, CgA is present in atrial myoendocrine, and conduction cells. In the human ventricular myocardium, it co-localizes with Natriuretic Peptide type B. The characterization CgA-derived peptides as intracardiac modulators, carried out in our laboratory has contributed to expand the concept of the endocrine heart. We found, by ex vivo functional studies, that through the action of its amino terminal (vasostatin) and catestatin domains, CgA exerts a direct cardiodepressive, antiadrenergic and cardioprotective influence, acting as a cardiac stabilizer under normal conditions and in the presence of stress (i.e. adrenergic, endothelinergic, and ischemia/reperfusion). At the same time, through the C-terminal serpinin, CgA elicits a beta-adrenergic-like cardiostimulatory effect. Cardioprotective effects of catestatin and serpinin occur also in spontaneously hypertensive rat hearts. Pasqua et al. (2013) documented that the intracardiac CgA processing is affected by physical (heart perfusion) and/or chemical stimuli (Isoproterenol and ET-1); the full-length CgA directly affects myocardial and coronary function by NO-cGMP-PKG cascade. Very recently, catestatin was proposed as pro-vascularization agent in “NO-OPTION” patients. However, the very short half-life and the susceptibility to degradation limit its clinical application. To overcome this problem, Angotti et al (2016) protected catestatin via encapsulation within Fibronectin-coated pharmacologically active microcarriers. Under these conditions, the peptide is released in a prolonged manner and is preserved from rapid degradation, representing a promising tool for therapeutic purpose. The above data support a multifaceted role of CgA and its derived peptides in cardiovascular homeostasis. The influence elicited on the heart under physio-pathological conditions, as well as the implication in the cardiac neuroendocrine scenario, is under intense investigation.

11:15 Session 5 Sumana Mahata

Chromogranin A regulates insulin storage and secretion as well as mitochondrial dynamics

Chromogranin A (CgA) has been implicated in the initiation and regulation of granule biogenesis and the sequestration of hormones in neuroendocrine cells. CgA still holds many enigmas with regard to its functions. In β-cell of the endocrine pancreas, CgA is a major cargo in insulin secretory vesicles. Here, we report the impact of CgA deficiency on the storage and secretion of insulin.

CgA is processed to vasostatin- and catestatin-containing fragments in pancreatic islet cells. Islets from CgA knockout (Chga-KO) mice display compensatory overexpression of chromogranin B, secretogranin II, SNARE proteins, and insulin genes as well as increased insulin protein content. At the ultrastructural level, Chga-KO β-cells contain fewer immature secretory granules than WT control but increased numbers of mature secretory granules (MSG), indicating an effect of CgA on vesicle maturation. We observed increased expression of multiple SNARE proteins involved in exocytosis, including Vamp1, Stx1a and Snap25. The increased SNARE protein expression correlated with a greater number of docked MSG at the plasma membrane coupled with exocytosis in Chga-KO mice. At the subcellular level, Chga-KO islets showed longer linear mitochondria and tubular networks, implicating mitochondrial fusion. In addition, compared to WT control, CgA-deficient β-cells exhibited increases in mitochondrial volume and numerical densities, as well as increased expression of nuclear encoded genes (Ndufa9, Ndufs8, Cyc1, and Atp5o). These changes in secretory vesicles and the mitochondria likely contribute to the increased glucose-stimulated insulin secretion (GSIS) observed in Chga-KO mice.

We conclude that CgA deficiency in pancreatic islets leads to (i) increased intracellular insulin levels accompanied with increased expression of the Ins1 and Ins2 genes, (ii) increased packaging and docking of insulin MSG, and (iii) elongated mitochondria owing to increased fusion coupled with increased GSIS, establishing a novel role of CgA in pancreatic biology and physiology.

#The Noland Scholarship from Caltech supported Sumana Mahata

Cytoskeleton and Organelle Dynamics
Chaired by Prof Luis M Gutierrez
11:30 Session 6 Luis Miguel Gutierrez

F-actin functions in the transport and organization of organelles in chromaffin cells

The F-actin cortical cytoskeleton is the major structure supporting the cell shape in chromaffin cells, and, in addition to this structural function, plays an essential role in multiples aspects of the secretory process. F-actin and microtubules cooperate to transport chromaffin granules from the internal region adjacent to the nucleus to the cell periphery where accumulate to replenish the vesicle secretory reservoirs. The retention of granules in the cortical F-actin structure causes the binomial distribution of the vesicles between cortical and internal populations, and this happen also to other organelles such mitochondria. In addition, mitochondria seem to adapt to the spaces formed by the F-actin cortical meshwork reducing its size when compared to that of the internal mitochondria.

Interestingly, F-actin structure is markedly different in cultured cells and the cells present in the bovine adrenal medulla. Thus in the “native” cell the F-actin in abundant both in the cortex and internal areas whereas in cultured cells this structure accumulates in the cortical area and is almost absent in the cell interior. In consequence, organelles and some proteins distribute differently in cultured and “native” cells, and this, in turn, could affect the influence of these organelles over the secretory dynamics.

This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness (MEC, Fondos FEDER: BFU2015-63684-P) to LMG.

11:45 Session 6 Sylvette Chasserot-Golaz

Actin cytoskeleton and Annexin A2:Essential Partners to form exocytotic sites in neuroendocrine cells

Calcium-regulated exocytosis results in the release of molecules such as neurotransmitters contained in secretory granules. In neuroendocrine cells, the recruitment and subsequent fusion of secretory granules at the plasma membrane occur at specific sites dedicated to exocytosis. Annexin A2 (AnxA2) was the first protein identified at exocytotic sites in chromaffin cells. AnxA2 binds two major actors of exocytosis, actin and phospholipids and mediates the formation of lipid microdomains required for the spatial organisation of fusion sites at the plasma membrane. To understand how AnxA2 promotes this membrane remodelling, the involvement of actin filaments in lipid domain organisation was investigated. Electron tomography of chromaffin cell revealed three distinct types of actin structures including a meshwork of actin filaments that paralleled the plasma membrane, but also large bundles of actin filaments connecting secretory granules to the plasma membrane and a coat of actin directly on the surface of secretory granules. The role of each of these actin structures observed at the granule docking sites remain unknown. However amperometric measurements of catecholamine release of chromaffin cells expressing AnxA2 mutant with impaired actin filament-bundling activity showed that cortical actin bundled by AnxA2 contributes to the formation of GM1-enriched lipid microdomains involved in the the docking and fusion of secretory granule at the plasma membrane (Gabel et al., 2015).

Numerous phosphorylation sites are present in the N-terminal domain of AnxA2, in particular the tyrosine 23 (Tyr23) whose phosphorylation affects the binding of AnxA2 to actin filaments and plasma membrane. We observed that nicotine stimulation triggers the phosphorylation of AnxA2 on Tyr23 at exocytotic sites. The expression of two AnxA2 mutants carrying phosphorylation deficient (Y23A) or phosphomimetic mutation (Y23E) reduced the number exocytotic sites. The expression of mutant AnxA2-Y23A inhibited the formation of lipid microdomains, whereas that of the AnxA2-Y23E affected the actin filaments associated with docked granule. Altogether, these results suggest that AnxA2 and the actin cytoskeleton are essential partners for granule docking and fusion and that Tyr23 phosphorylation of AnxA2 is critical for exocytosis.

12:00 Session 6 Ana Cárdenas

Cortactin controls the exocytotic fusion pore by a mechanism that involves remodeling of actin filaments.

Cortactin is an actin-binding protein that promotes actin polymerization in synergy with the nucleation promoting factor N-WASP. In the present work we examined the role of cortactin in the Ca2+-induced formation of actin filaments and exocytosis. With this aim we expressed in bovine chromaffin cells different cortactin domains or mutants enable to interact with proline-rich domain (PRD)-containing proteins, like including N-WASP, or to be phosphorylated by Ca2+-dependent kinases, such as ERK1/2 or and Src. Our results show that the activation of nicotinic receptors in chromaffin cells promotes cortactin translocation to the cell cortex, where it colocalizes with actin filaments. We further found that, in association with PRD-containing proteins, cortactin contributes to the Ca2+-induced actin filament formation and to regulate fusion pore dynamics and number of exocytotic events induced by activation of nicotinic receptors. However, whereas the actions of cortactin on fusion pore dynamics depend on the availability of monomeric actin and cortactin phosphorylation by ERK1/2 and Src kinases, this actin binding protein regulates the extent of exocytosis by a mechanism independent of actin polymerization, and that is determined by its phosphorylation by ERK1/2. Together our findings point out a role for cortactin as a critical modulator of actin filament formation and exocytosis in neuroendocrine cells. This work has been supported by the grants FONDECYT 1160495 and P09-022-F from ICM-ECONOMIA, Chile.

12:15 Session 6 Frederic Meunier

The “casting net” hypothesis: vesicular capture, translocation to the plasma membrane and consequences on the nanoscale organization of fusion sites

In this presentation, I will examine the role of the cortical actin network in the final steps leading secretory vesicles to dock and fuse with the plasma membrane of neurosecretory cells thereby releasing neurotransmitter and hormones. Following an active transport to the periphery of the cell, secretory vesicles are recruited in a Ca 2+ - dependent manner to the cortical actin network via the anchoring action of Myosin VI short insert (1). Upon stimulation, the cortical actin network undergoes a Myosin II- dependent relaxation that culminate in pulling bound secretory vesicles towards the plasma membrane (2). The analogy of the cortical actin network acting as a casting net dragging vesicles toward the plasma membrane provides a plausible hypothesis to explain the synchronous translocation of secretory vesicles occurring upon secretagogue stimulation. I will also discuss the consequences of the cortical actin network approaching the plasma membrane on the nanoscale organization and mobility of key proteins of the exocytic machinery (3, 4).

12:30 Session 6 Gerardo J. Félix-Martínez

Modeling the effect of organelle distribution on the secretory response of bovine chromaffin cells

Secretion from chromaffin cells is a Ca2+-dependent process that relies on the formation high- Ca2+ microdomains (HCMDs) in the vicinity of the exocytotic machinery. It has been proposed that functional triads composed of Ca2+ channels, endoplasmic reticulum (ER) and mitochondria heavily contribute to the formation of HCMDs. Recent experimental observations have demonstrated that ER and mitochondria are not homogeneously distributed throughout the cytosol of bovine chromaffin cells. Instead, it was shown that subpopulations of both organelles are located at the cortical region of the cell, where both Ca2+ channels and exocytotic sites are also located. In this work, we developed a stochastic three-dimensional model to evaluate the effect of a non-uniform distribution of cortical organelles on the formation of local HCMDs near a cluster of Ca2+ channels and, therefore, on the secretory activity of bovine chromaffin cells. Our simulations show that the presence of cortical organelles in the vecinity of the Ca2+ channels produces an increase in the local concentration of Ca2+ as a consequence of both the amplification effect of the Ca2+-induced Ca2+-release mechanism from the ER and the organelles acting as physical barriers to Ca2+ diffusion. In addition, our model suggests that this increase in local Ca2+ enhances the secretory response of the cell as indicated by the faster exocytotic dynamics predicted by our simulations. In addition, according to our simulations, cortical organelles could affect the spreading of the Ca2+ signal and, as a result, the secretion of non-colocalized vesicles located far away from the Ca2+ channels. Finally, our model also predicts that the location of the ryanodine receptor channels with respect to the other mechanisms involved in the regulation of Ca2+ in the subplasmalemma region of the cell could play an important part in the fine tuning of the secretory response of the cell.

12:45 LUNCH
14:00 Plenary 5 Annette Dolphin

Neuronal calcium channel trafficking and function: relevance to chronic pain.


New Insights into ion channels controlling stimulus-secretion coupling in chromaffin cells
Chaired by Prof E Carbone
15:00 Session 7a Emilio Carbone

Na+, Ca2+, and K+ channels generating repetitive firing and rhythmic bursting in mouse chromaffin cells

Adrenal chromaffin cells (CCs) are the main source of circulating catecholamines (CAs) that regulate the body response to stress. Release of CAs is controlled neurogenically by the activity of preganglionic sympathetic neurons through action potential (AP) trains. APs in CCs are generated by robust depolarization following the activation of nicotinic and muscarinic receptors that are highly expressed in CCs. Bovine, rat and mouse CCs also express a composite array of Na+, K+ and Ca2+ channels that regulate the resting potential, shape the APs and set the frequency of AP trains. If chromaffin cells simply relay preganglionic nerve commands into AP trains that induce CAs release, then why these cells need so many ion channels to produce regular AP trains? Recent observations have shown that ion channel complexity may derive from the ability of CCs to undergo complex firing patterns that derive from an intrinsic CCs excitability, non-neurogenically controlled. We have recently shown that CCs can sustain persistent burst firing when cultured mouse CCs are steadily depolarized (Vandael at al, 2015) or acidic pHo induces the block of pH-sensitive TASK-1 and Ca2+-dependent BK channels (Guarina et al., 2017). Similar burst firings are observed in mouse CCs of adrenal gland slices in which the BK-β2 subunit, responsible for the fast inactivation of BK channels, is genetically deleted (Martinez-Espinosa et al, 2014). Here, we will discuss what causes burst firings in CCs, trying to give a rationale to the role of Nav and Cav channels that support repeated AP firing and sustained bursts by balancing their contribution with the many K+ channel types (Kv1-4, Kv7, BK, SK, TASK, ERG) expressed by CCs.

15:15 Session 7a Iago Mendez

Muscarinic receptors activation either blocks spontaneous firing activity or causes rhythmic bursting in resting mouse chromaffin cells

Mouse chromaffin cells (MCCs) exhibit spontaneous slow action potential (AP) firing activity (≈ 1 Hz) at rest, which is converted into sustained firing of higher frequency (5-15 Hz) during step depolarization. MCCs firing can undergo either repetitive or burst firing patterns depending on the degree of cell depolarization, Nav channel availability, co-expression of BK channel β-subunits and acidic pHo (Lingle C, Martinez-Espinosa PL, Guarina L & Carbone E, 2017; in press). Since muscarinic acetylcholine receptor (mAChR) activation is known to produce sustained CCs depolarization it is interesting to test whether mAChR acts on cell activity by inducing burst firing.

Here we report that 30 s to 1 min continuous perfusion of either muscarine (Mus; 30 μM) or oxotremorine M (Oxo-M; 10-30 μM) have variable effects on MCCs AP firing. In 60% of resting MCCs (n= 28), Mus or Oxo-M have excitatory effects while in the remaining 40% the two agonists have either no action or mild hyperpolarizing effects that may silence spontaneously active cells. The excitatory response is accompanied by a robust cell depolarization (5-10 mV) followed by an increased firing frequency that is often converted in sustained bursts. In these cells, application of Musc or Oxo-M in voltage clamp conditions (-50 mV) induce mean inward currents of ≈5 pA, suggesting that mAChR agonists act by blocking a resting K+ channel conductance. Similar effects are observed by applying the M-current (Kv7) selective blocker (XE991; 3 μM). Voltage-clamp protocols that uncover the slow closing kinetics of Kv7 show that this current is blocked by muscarine in a less percentage of cases than XE991. This suggests that muscarinic effects that produce burst firing proceeds not only through the block of Kv7 channels t but may involve the block of other K+ channels.

15:30 Session 7a Masumi Inoue

Molecular identity and functions of TASK1-like channels in adrenal medullary cells

Muscarinic receptor stimulation and a decrease in extracellular pH induce catecholamine secretion through inhibiting TWIK-related acid-sensitive K+ 1 (TASK1)-like channels in rat adrenal medullary (AM) cells. To elucidate the molecular identity of TASK1-like channels in mouse AM cells, the perforated patch clamp technique was used to examine the effects of gene deletion on the whole-cell current. The inward current reversibly developed in response to step changes in external pH from 7.4 to 6.0 in wild-type mouse AM cells. The amplitudes of the acidosis-induced current were well approximated by a logistic equation with IC50 of pH7.07. This pH-dependent production of current was abolished by the genetic depletion of TASK1, but not TASK3. However, the holding current level at -50 mV and the slope conductance in the voltage range from -50 mV to -70 mV did not significantly differ between in wild-type and TASK1-knockout (KO) AM cells. Immunocytochemistry revealed that TASK1-like immunoreactivity (IR) was mainly present at the cell periphery in wild-type AM cells, but not in TASK1-KO. TASK3-like IR was rarely detected in wild-type AM cells and not enhanced in TASK1-KO, suggesting that loss of TASK1 was not compensated by upregulation of TASK3. A decrease in external solution induced catecholamine secretion in wild-type AM cells, but not TASK1-KO. The muscarine-induced current in wild-type AM cells consist of quinine-sensitive and insensitive components and the latter was abolished in 73% of the cells examined by the deletion of TASK1 gene. These results indicated that TASK1 channels are suppressed by a decrease in external pH or muscarinic receptor stimulation in mouse AM cells.

15:45 Session 7a Arturo Picones

Native Ionic Currents Recorded from Primary Cultured Chromaffin Cells by Automated High-Throughput Patch-Clamp Electrophysiology

The application of automated patch-clamp (APC) technology and instrumentation has made a profound impact on strategies for the discovery and development of new drugs targeting ion channels. APC instrumentation has proven to increase data production in at least one order of magnitude, maintaining the high quality of ion current measurement under voltage clamp and Gigaseal (GΩ) requirements. Significantly relevant for the expansion of APC technology is to extend its success to the analysis of endogenous ion channels expressed in native and primary cells. Here we present the optimization and validation of the recording of endogenous ion currents from native neuroendocrine chromaffin cells in primary culture utilizing an APC system. Cells were enzymatically dissociated from bovine adrenal glands freshly obtained from recently slaughtered adult cattle (2 to 3 years of age); maintained for 2–4 days with DMEM supplemented with Ham's F12, 10% fetal bovine serum and Penicillin-Streptomycin 1%, at 37°C and 5% CO2. Then, cells were detached by TrypLE Express (3 min), centrifuged and resuspended to a cell density of 3 million/ml. Whole-cell automated patch-clamp experiments were performed on a QPatch 16X instrument (Sophion Bioscience), while classic manual patch-clamp whole-cell recordings were carried out with an Axopatch 200B/Digidata 1550/pCamp10 (Molecular Devices). Results obtained with the APC system showed a Na+ inward current and two outward K+ currents which in all aspects were indistinguishable from currents recorded by classic manual patch-clamp from chromaffin cells from the same batches. The overall completed-experiment success rate was 54%. Their characteristic kinetics, voltage dependence and pharmacology correspond to Na+ and K+ (SK and BK) permeable channels, already identified and well characterized in the bovine chromaffin cells literature. These results validate the use of APC technology for the study of this neurosecretory cellular type which complies with the essential requirements of yielding dissociated cells in sufficient high density and easily identifiable ion channel activity.

Supported by SECITI 039/2013, CONACYT grants 279820 (LaNCa), CB 240305 and PAPIIT-UNAM IN211616.

16:00 Session 7a Arturo Hernandez Cruz

The role of GABA A receptor activation on rat chromaffin cells in situ.

The role of GABA in adrenal medulla chromaffin cell (CC) function is just beginning to unfold. GABA is stored in catecholamine (CA)-containing dense core granules and it is probably released together with CA, ATP and opioids in response to physiological stimuli, playing an autocrine-paracrine role in CCs, by interacting with ionotropic GABAA receptors (GABAA-R). The paradoxical "dual action" of GABAA-R activation: enhancement of CA secretion and inhibition of synaptically evoked CA release appears to be only one aspect of GABA´s multifaceted actions. In this review we discuss recent physiological experiments on rat CCs in situ showing that GABA regulates CCs function differently depending on its rate of presentation: During non-stressful conditions and slight cholinergic transmission, GABAA-Rs activation by endogenous GABA inhibits spontaneous ACh release from splanchnic nerve terminals and decreases spontaneous Ca2+ fluctuations preventing unnecessary CA secretion. During acute stress, intense stimulation of splanchnic cholinergic nerve terminals causes strong depolarization and bursts of action potentials in CCs, allowing the Ca2+ influx required for a vigorous CA release. However, CA exocytosis decays by voltage-independent inhibition of L-type Ca2+ channels by signalling cascades and rapid desensitization of cholinergic nicotinic receptors. Membrane depolarization induced by fast GABAA-R activation may help to maintain continuous CCs stimulation and CA exocytosis under intense stress. GABAAR activation is not excitatory in about 50% of the rat CCs population in situ, either because it hyperpolarizes then or elicits no visible response. The proportion of CCs responding to GABA one way or another varies depending on the intracellular chloride concentration ([Cl-]i). The role of the main transporters NKCC1 and KCC2, which accumulate or extrude Cl-, respectively in regulating [Cl-]i in CCs is also discussed. These findings emphasize a novel and complex GABA-mediated regulatory mechanism on CCs activity and hence, on the control of CA secretion.

Receptor regulation of catecholamine secretion
Chaired by Prof Sun
16:30 Session 7b Jin-Peng Sun

G protein independent, Arrestin biased GPCR agonism induces acute catecholamine secretion through TRPC3 coupling

Acute hormone secretion triggered by G protein-coupled receptor (GPCR) activationunderlies many fundamental physiological processes. In the conventional paradigm, thereceptor-G protein complex initiates such “first-wave signaling” events. With the exception ofdesensitization, the receptor-arrestin complex induces signaling that normally occurs later,called “second-wave signaling.” Here, we revealed that β-arrestin-1- biased agonismstimulates acute catecholamine secretion through transient receptor potential cation channelsubfamily C (TRPC3) coupling. The β-arrestin-biased agonist promoted the recruitment ofTRPC3 or phosphoinositide-specific phospholipase C (PLCγ) to the angiotensin II receptortype 1 (AT1R)-β-arrestin-1 signaling complex. Replacing the C-terminal region of β-arrestin-1 with its counterpart β-arrestin-2 or using a specific TAT-P1 peptide to block the interactionbetween β-arrestin-1 and PLCγ abolished TRV120027-induced TRPC3 activation. Thereceptor-β-arrestin-1- TRPC3 signaling constitutes an important component after theactivation of endogenous AT1R or muscarinic acetylcholine receptors, which may occur inclinical or physiological conditions, such as hypertension or parasympathetic nerve activation.Taken together, the GPCR-arrestin complex initiated non-desensitized “first-wave” signalingnot only in endosomes but also on the plasma membrane through fast communication to ionchannels could be a general mechanism in many cellular processes.

17:00 Session 7b Xiao Yu

Regulation of agonist induced catecholamine secretion from chromaffin cells by a tyrosine phosphatase

G protein coupled receptors are the largest family of membrane proteins and_x000D_ the most important targets of clinical drugs. Activation of GPCR play key roles_x000D_ in catecholamine secretion from chromaffin cells isolated from adrenal medulla._x000D_ Downstream of GPCR, an explicit network governs the time course and extent of_x000D_ catecholamine secretion. Here we identified that a tyrosine phosphatase is_x000D_ important in regulation of agonist induced catecholamine secretion using specific_x000D_ phosphatase inhibitors. The electrophysiology and dynamic analysis indicated_x000D_ that this tyrosine phosphatase regulated catecholamine secretion by controlling_x000D_ the phosphorylation states of the receptor and several important downstream_x000D_ effectors. Moreover, crystal structure of the phosphatase in complex with its_x000D_ substrate indicated that key residues in the WPD loop and Q loop are important_x000D_ for its substrate recognition. Further in vivo functional studies confirmed the_x000D_ relevance of these key residues of the phosphatase in contribution of its_x000D_ regulatory role in agonist induced catecholamine secretion from chromaffin cells._x000D_ Taken together, we have identified that a specific tyrosine phosphatase plays_x000D_ important roles in regulation of the delicate network of chromaffin cells to_x000D_ control catecholamine secretion and provided its underlying structural basis by_x000D_ crystallographic and enzymology studies.

17:15 Session 7b Jiangping Wu

Receptor tyrosine kinase EPHB6 and testosterone regulate catecholamine synthesis and release in adrenal gland chromaffin cells.

EPHB6 belongs to the largest EPH receptor tyrosine kinase family. It acts on two types of cells: vascular smooth muscle cells (VSMCs) and adrenal gland chromaffin cells (AGCCs). EPHB6 deletion results in increased VSMC contractility but reduced 24-h urine catecholamine (CAT) levels, which increase to the normal range after castration while VSMC contractility remains elevated. As a result, castrated KO mice have increased blood pressure. Mechanistically, male KO AGCCs presented defective Ca2+ influx caused by a larger BK current, which leads to early closure of voltage-gated calcium channels.

In this study, we examined CAT synthesis in KO AGCCs. The male KO adrenal glands presented reduced total CAT content. The expression of the rate-limiting enzyme tyrosine hydroxylase (TH) for CAT synthesis in male KO AGCCs was significantly lower. DNA microarray, followed by RT-qPCR and immunoblotting, revealed that a transcription factor Egr-1 expression was reduced in male KO AGCCs. Egr-1 acts on an enhancer of the TH gene, and its knockdown by siRNA reduced TH expression and CAT synthesis. Therefore, EPHB6 regulates Egr-1 expression, which controls TH synthesis; the TH levels in turn determine the CAT synthesis. This explains the defective CAT synthesis in EPHB6 KO AGCCs.

The CAT release in KO AGCCs was next assessed. Electron microscopy (EM) showed that male KO AGCCs had smaller immediately releasable pool of CAT-containing vesicles. Consistent with the EM finding, amperometry recordings demonstrated that male KO AGCCS had significantly lower spikes per cell in a 60-sec period and reduced charge per spike and per feet after cholinergic stimulation, suggesting smaller numbers of vesicles and less CAT content per cell released after cholinergic stimulation. The male KO cells also showed less F-acting disintegration after the stimulation, preventing CAT-containing vesicles to migrate to the cell membrane. This likely explains the reduced CAT release by male KO AGCCs after cholinergic stimulation. The above-described defects in male KO AGCCs related to CAT synthesis and release were corrected after castration, indicating that the effect of EPHB6 deletion in AGCCs depended on the presence of testosterone.

Our human genetic study in 1500 hypogonadic male patients showed that a SNP in a major acetyl choline receptor (AchR) subunit 3 gene was significantly associated with hypertension risk. AchR is a molecule from which the signals for CAT synthesis and release are started. Indeed, we further demonstrated that EPHB6 was physically associated with the AchR 3 subunit.

Our study revealed that EPHB6 and testosterone in concert regulate CAT synthesis and release, which in turn modulate blood pressure. For those males with EPHB6 loss-of-function mutations, hypogonadism due to ageing or some medical conditions will increase their hypertension risks. For these patients, testosterone replacement therapy could be a safe and effective treatment for hypertension.

17:30-18:00 Session 7b Almudena Albillos

Characterization and pharmacology of nicotinic receptors in human chromaffin cells.

During the last 10 years we have been working on human chromaffin cells obtained from the adrenal gland of organ donors that suffered encephalic or cardiac death. We first electrophysiologically characterized the nicotinic acetylcholine receptors (nAChRs) activated by acetylcholine, and their contribution to the exocytosis of chromaffin vesicles and release of catecholamines. We have shown that these cells possess an adrenergic phenotype. This phenotype may contribute to an increased expression of α7 nAChRs in these cells, allowing for patch-clamp electrophysiological recording of α7 nAChR currents, something that had previously not been achieved in non-human species. We also characterized non-α7 nAChR subtypes by means of the use of α-conotoxins in electrophysiological experiments and, together with molecular biology experiments, conclude that the predominant nAChR subtype in human chromaffin cells is α3β4* (asterisk indicates the posible presence of additional subunits). In addition, there is a minor population of αxβ2 nAChRs. Both α7 and non-α7 nAChR subtypes contribute to the exocytotic process. Exocytosis mediated by nAChRs could be as large in magnitude as that elicited by calcium entry through voltage dependent calcium channels. Finally, we have also investigated the effect of nAChR-targeted tobacco cessation drugs on exocytosis in chromaffin cells. We have concluded that at therapeutic concentrations, varenicline alone does not increase the frequency of action potentials evoked by ACh. However, varenicline in the presence of nicotine does increase this frequency, and thus, in the presence of both drugs, the probability of increased catecholamine release in human chromaffin cells is high.

19:00 Conference dinner

Saturday 26th August

Session number
Chromaffin Cells in Stress
09:00 Nathalie C. Guerineau

Stress-induced remodeling of adrenal stimulus-secretion coupling: evidence for bidirectional adaptive mechanisms.

In mammals, catecholamine secretion from adrenal chromaffin cells represents an ubiquitous mechanism helping the organism to cope with stress. Once delivered into the blood circulation, epinephrine and norepinephrine exert multiple actions, leading to physiological adjustments enabling the organism to cope with a threat for its survival. While an instantaneous secretion of catecholamines is beneficial, repeated or prolonged stressful situations are detrimental and can initiate many diseases. Adrenal catecholamine secretion relies on a neurogenic command arising from the splanchnic nerve terminals synapsing onto chromaffin cells and a local gap junctional communication. In response to a systemic stress (5 day-cold exposure, male Wistar rats), both cholinergic synaptic transmission and gap junctional coupling are remodeled. The frequency of excitatory postsynaptic events is increased and the respective contribution of alpha3- and alpha9-containing nAChRs to a cholinergic challenge is modified. While alpha9 nAChRs dominantly support acetylcholine-induced current in cold-stressed rats, alpha3 nAChRs are the main contributing channels in unstressed animals. Expression of Cx36 and Cx43 gap junctions between chromaffin cells is upregulated, leading numerous cells to exhibit co-active Ca2+ signals upon nicotinic application. All these functional changes contribute to the increased plasma catecholamine levels in cold-stressed rats. We next examined the plasticity of the stimulus-secretion coupling in rats suffered from arterial hypertension, a stress-related pathology. Upon robust depolarization, chromaffin cells of adult spontaneously hypertensive male rats are less excitable than cells from normotensive rats. Postsynaptic events also undergo remodeling, as evidenced by a reduced frequency in response to a depolarizing challenge and changes in expression of transcripts encoding nAChR subunits. Additionally, gap junction coupling between chromaffin cells is reduced. These changes are associated with a reduced catecholamine secretion evoked by a robust cholinergic stimulation. As such, the adrenal medullary tissue dualistically and accurately adapts the competence of the stimulus-secretion coupling to appropriately manage catecholamine secretion.

09:30 Lee Eiden

PACAP signaling in stress: Insights from the chromaffin cell.

The pituitary adenylate cyclase-activating polypeptide (PACAP) was identified by Miyata and Arimura in 1989, as a 38-amino acid neuropeptide present in extracts of the ovine hypothalamus, causing elevation of cyclic AMP in perfused hemi-pituitary glands. PACAP acts at three distinct receptors PAC1, VPAC1, and VPAC2 (the last two also activated by the related peptide VIP) all coupled to adenylate cyclase activation through Gs. PACAP has been identified as the adrenomedullary neurotransmitter in stress through a combination of ex vivo, in vivo, and in cellula experiments over the past two decades. The mechanisms whereby PACAP causes catecholamine secretion, and the activation of catecholamine biosynthetic enzymes during episodes of stress in mammals, has been extensively studied in the chromaffin cell. Features of PACAP signaling to the chromaffin cell allowing stress transduction at the adrenal medulla have yielded insights into the contrasting roles of acetylcholine and PACAP action as first messengers at the sympathoadrenal synapse, via differential release by different patterns of splanchnic nerve firing, and dramatically different signaling pathways leading to catecholamine secretion and chromaffin cell gene transcription. The discovery of activation of the novel cAMP sensor NCS-Rapgef2, for example, occurred in the chromaffin cell. The ability of PACAP to cause catecholamine secretion via calcium influx independent of ionotropic channel activation and action potential generation remains a focus of active investigation in several laboratories, both at the chromaffin cell, and within autonomic ganglia of both the parasympathetic and sympathetic nervous systems. PACAP is a neurotransmitter important in stress transduction in the central nervous system as well, and is found at stress-transduction nuclei in brain including the paraventricular nucleus of hypothalamus, the amygdala and extended amygdalar nuclei, and the prefrontal cortex. The current status of PACAP as a ‘master regulator’ of stress signaling in the nervous system derives fundamentally from establishment of its role as the adrenomedullary transmitter in stress, and experimental elucidation of PACAP action at this synapse remains at the forefront of understanding its role in stress signaling throughout the nervous system.

Disease causing mutations affecting exo-endocytosis
Chaired by Antonio Garcia
10:30 Session 8 Michael Cousin

Loss of functional huntingtin causes activity-dependent presynaptic defects in Huntington’s disease.

Huntington’s disease (HD) is a monogenic degenerative condition caused by a CAG expansion in exon 1 of the gene encoding huntingtin. In a number of neurodegenerative conditions dysfunctional neurotransmission can lead to synaptic failure and loss. In HD, synaptic atrophy is particularly prevalent in striatal medium spiny neurons. We tested the hypothesis that their vulnerability arises from an inability to sustain performance during intense neuronal activity in primary neuronal cultures derived from a mouse model of HD (httQ140/Q140 knockin mouse). These studies revealed two distinct activity-dependent signatures of presynaptic dysfunction. First, we discovered that activity-dependent bulk endocytosis, which is only triggered during elevated neuronal activity, was increased in HD neurons derived from different brain regions. Second, we uncovered a retardation in clathrin-mediated endocytosis which was specific to striatal neurones and only occurred during elevated activity. Both activity-dependent disease signatures are observed in neurons that have one mutant huntingtin allele (+/httQ140), reflecting the disease condition. Depletion of endogenous huntingtin from wild-type neurons recapitulated both disease signatures, whereas depletion of mutant huntingtin in HD neurons had no effect. Therefore loss of wild-type huntingtin function is responsible for both activity-dependent disease signatures. This was confirmed when presynaptic dysfunction was rescued by the expression of wild-type huntingtin in HD neurons. The intrinsic susceptibility of specific subtypes of HD neurons to elevated neuronal activity could therefore render them progressively vulnerable to ongoing physiological firing patterns, potentially explaining synapse failure and degeneration in later life.

11:00 Session 8 Antonio Garcia

Altered single-vesicle exocytosis in chromaffin cells from mouse models of neurodegenerative disease.

Altered synaptic dysfunctions have been observed in various neurodegenerative diseases. These have been studied mostly in central synapses, monitoring transmitter release through postsynaptic excitatory or inhibitory currents or cytosolic Ca2+ signals. Surprisingly, a few studies have also reported changes in the kinetics of exocytosis in adrenal chromaffin cells (CCs) from various transgenic mouse models of neurodegenerative diseases.

Chronologically, these studies are as follow: (1) Parkinson’s disease (PD) related protein a-synuclein, overexpressed in mouse CCs (MCCs) inhibits a vesicle priming step (Larsen et al., 2006; J. Neurosci. 26: 11915); (2) single vesicle secretion from R6/2 mouse model of Huntington’s disease (HD) was decreased because vesicles had a smaller quantal size (Johnson et al., 2007; J. Neurochem. 103, 2102); (3) faster kinetics but smaller quantal size of single-vesicle exocytosis occurs in CCs from APP/PS1 mouse model of Alzheimer’s disease (AD) (De Diego et al. 2012; Biochem. Biophys. Res. Commun. 428, 482); (4) Huntingtin associated protein 1 (HAP1) is endogenously expressed in MCCs, controlling vesicle docking, the readily releasable pool size and fusion pore stability (McKenzie et al. 2014. J. Physiol. 592, 105); (5) in the SOD1/G93A mouse model of amyotrophic lateral sclerosis (ALS), the single-vesicle exocytotic kinetics was slower but with higher quantal size (Calvo-Gallardo et al. 2015. Am. J. Physiol. 308, C1); and (6) in the R6/1 mouse model of HD we have found drastic changes in CC excitability, ion currents, and fusion pore kinetics (unpublished).

These accumulating changes of exocytosis occurring in CC carrying mutations or expressing proteins linked to PD, AD, HD, and ALS raise at least two hypothesis: 1) those changes are directly linked to disease pathology; 2) the changes are secondary to stress conditions affecting the sympathoadrenal axis.

We predict a growing interest in defining these and other changes in adrenal CCs of various mouse models, carrying various mutations linked to human familiar neurodegenerative diseases.

11:30 Session 8 Emanuele Sher

Calcium- and SNARE-dependent release of pathological Tau from rodent and human AD synaptosomes.


12:00 Session 8 Carmen Nanclares

Alterations in excitation-secretion coupling in chromaffin cells related to the progression of Alzheimer´s disease in 3xTg-AD transgenic mice.

Alzheimer´s disease (AD) is the most common form of dementia. The alteration of several neurotransmitter systems has been reported. These alterations in the neurotransmission processes could be correlated with changes in the synthesis, storage or release of neurotransmitter.

In this study we have used a triple transgenic murine model of AD (3xTg-AD). This animal model contains mutations in the gene encoding the amyloid precursor protein (βAPPSwe), presenilin-1 (PS1M146V) and tauP301L, which mimics the development of the disease on Alzheimer's patients. We propose to study here the last steps of exocytosis in chromaffin cells of 3xTg-AD mice of different ages using the amperometric technique. We have found significant changes in the exocytosis of catecholamines occurring in mice of 6 and more than 12 months of age, where the pathology is already established, when compared with prepathological mice (2 months). These changes show an increase of the number of amperometric spikes during the development of the disease, although the quantal catecholamine content of each spike is lower. Kinetic analysis of secretory spikes shows that as the disease progresses amperometric spikes are faster in triggering and shorter in duration. Patch-clamp technique was also used to measure the different ionic currents involved in the physiological release of catecholamines. We found an increase in all these ionic currents, namely, nicotinic, sodium, calcium and potassium currents. Membrane potential measurements have also been performed, finding it slightly hyperpolarized in chromaffin cells of mice older than 12 months of age, resulting in a greater depolarization against a stimulus with ACh. Taken together, these findings suggest that throughout the development of 3xTg-AD mice and as Alzheimer's disease is established there is a change in chromaffin cell excitability, which causes neurotransmission to accelerate. These alterations could have an impact on the response that the organism offers in a stressful situation.

12:15 Session 8 Ye Jin Chai

Munc18-1 controls α-synuclein self-replicating aggregation in Early Infantile Epileptic Encephalopathy.

Munc18-1 is an key regulator of the exocytic machinery. Munc18-1 heterozygous mutations are responsible for developmental defects, neurodegenerative and epileptic phenotypes including infantile epileptic encephalopathy (EIEE) and it is unclear whether the disease stems from a loss of function or a gain of pathological function. Here, we used single molecule analysis, gene-edited cells and neurons to demonstrate that Munc18-1 EIEE-causing mutations promote the formation of large polymers that co-aggregate wild-type Munc18-1 in vitro and when expressed in neurosecretory cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body-like ring structures that contain α-synuclein (α-Syn). We reveal that not only Munc18-1 binds α-Syn but its EIEE mutants also co-aggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-SynWT and that of Parkinson’s disease-causing α-SynA30P mutant, an effect rescued by Munc18WT expression indicative of chaperone activity. Co-expression of α-SynA30P mutant with Munc18-1WT reduced the size of α-SynA30P aggregates. Munc18-1 EIEE mutations may therefore lead to a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn. Our results uncovers an unexpected function of Munc18-1 in controlling α-Syn propensity to aggregate.

Poster Presentations

Poster No.
First Author
Poster Session
1 Michelle Juan-Bandini Who's first? The stimulus-secretion coupling according to the age of secretory granules 1
2 Jose David Machado Isolation of mouse chromaffin secretory vesicles 1
3 Judith Estevez Herrera Chromaffin adrenergic cells without adrenaline 1
4 Johan Dunevall Sub-Vesicular Distributions of Catecholamine Storage Revealed by Intracellular Vesicle Cytometry Recordings in Adrenal Chromaffin Cells. 1
5 Soodabeh Majdi Extracellular ATP regulates the vesicular content in chromaffin cells and increases the fraction released during individual exocytosis events 1
5 Soodabeh Majdi Extracellular ATP regulates the vesicular content in chromaffin cells and increases the fraction released during individual exocytosis events 1
6 Anna Larsson Exploring the mechanism of action of ATP on storage and release of catecholamines in chromaffin cells 1
7 Zuleirys Santana-Rodriguez Synaptotagmin Isoforms Confer Distinct Activation Kinetics and Dynamics to Dense Core Granules 1
8 Joana Martins Alternative splicing of Synaptotagmin-7 regulates large dense-core vesicle priming 1
9 Bassam Tawfik Synaptotagmin-7 cooperates with synaptotagmin-1 to ensure fast Ca2+-triggered exocytosis from adrenal chromaffin cells 1
10 Judit Meszaros Cells in pain: synaptotagmins controlling CGRP secretion in sensory neurons 1
11 Yolanda Gimenez Molina Doc2B as a calcium sensor protein linked to Munc13-1 dynamics and STIM contacts in chromaffin cells 1
12 Carmen Martínez Ramírez Opposite effects of ER Ca2+ handling on exocytosis in mouse and bovine chromaffin cells 1
13 Muhmmad Omar-Hmeadi Plasma membrane PI(4,5)P2 is critical for secretory granule exocytosis 1
14 Misty R Marshall Molecular mechanisms of v-SNARE function in secretory granule exocytosis. 1
15 Ying Zhao Estimating the number SNARE proteins changing conformation in a single fusion event 1
16 Qinghua Fang SNARE Complex Reporter 2: An Improved version of SNAP25 based FRET construct 1
17 Joannalyn Delacruz Narrow fusion pores in chromaffin cells are cation selective 1
18 Satyan Sharma SNARE mediated fusion pore – mechanism and nature 1
19 Lucas Bayonés Dynamin-Dependent and Independent Membrane Retrieval after Immediately Releasable Pool (IRP) Exocytosis 2
20 Montenegro Mauricio Norman Effect of Cytosolic Ca2+ and F-Actin Polymerization on Fast Endocytosis and Rapid Replenishment of Immediately Releasable Pool in Mouse Chromaffin Cells 2
21 Pika Miklavc The role of microtubule and actin cross-linking proteins for the post-fusion phase of exocytosis 2
22 Ana M Cardenas Altered cortical actin polymerization in dysferlin-deficient skeletal myocytes 2
23 Alexandre Milman A sodium-permeable background conductance finely tunes action potential firing of adrenal mouse chromaffin cells in situ: involvement of the sodium leak channel NALCN? 2
24 Ayoze Gonzalez-Santana Modulation of catecholamine exocytosis through incretin receptors 2
25 Alejandre-Garcia Tzitzitlini Modulation of membrane potential by GABAA receptors by exogenous and endogenous GABA in rat chromaffin cells recorded in acute adrenal slices 2
26 Alice Dallatomasina CgA1-373 and CgA1-372 interact with nicotinic acetylcholine receptors (nAChR) and regulate angiogenesis 2
27 Pena-Del Castillo Johanna Enhancement of Ca2+ release from intracellular stores and catecholamine hypersecretion precedes hypertension in adrenal chromaffin cells from spontaneously hypertensive rats 2
28 Stephen Bunn Interleukin-6 signalling in the mouse adrenal gland. 2
29 Iago Mendez Lopez Mitochondrial ultrastructure and function is impaired in chromaffin cells of the SOD1G93A mouse model of ALS and precede exocytotic alterations at a pre-disease stage 2
30 Andres M. BARAIBAR Alterations in the stimulus-secretion coupling related to aging in the murine model of accelerated senescence SAMP8 2