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Professor and Chair John Anagli, PhD Adjunct Assoc. Prof Rodrigo Andrade, Ph.D. Professor Jacob Aranda, MD, PhD Prof. Pediatrics, Pharm Cristina Artalejo, MD, PhD Assoc. Professor Michael Bannon, PhD Professor Julie Boerner, PhD Asst Prof. KCI, Pharm Chaya Brodie, PhD Adjunct Professor Dharam Chopra, PhD Prof. IEHS, Pharm Nicholas Davis, PhD Assoc. Prof. Gregory Kapatos Professor David Kessel, PhD Professor Thomas Kocarek, PhD Assoc. Prof. IEHS, Pharm Lawrence Lash, PhD Professor Karin List, PhD Asst. Prof Larry Matherly, PhD Professor Raymond Mattingly, PhD Assoc. Prof. Roy B. (Mac) McCauley, PhD Professor Kamiar Moin, PhD Subsidy Assoc.Prof. Raymond Novak, PhD Director IEHS, Pharm John Reiners, Jr., PhD Prof., IEHS, Pharm Sandra Rempel, PhD Adjunct Assoc Prof Tiziano Scarabelli, MD.PhD Adjunct Assoc Prof. David Schneider, PhD Assoc. Prof. Eugene Schoener, PhD Professor Robert B. Silver, PhD Prof. Pharm, Physiol, Radiol. Manuel Tancer, MD Prof. Psychiatry, Pharm Stanley Terlecky, PhD Assoc. Prof. Ellen Tisdale, PhD Assoc. Prof. Arun Wakade, PhD Professor Hai-Young Wu, PhD Assoc. Prof. Akio Yamazaki, PhD Prof. Ophthal., Pharm Russell Yamazaki, PhD Assoc. Prof, Assoc. Chair
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Cristina R. Artalejo,
M.D., Ph.D. My laboratory has embarked on an ambitious program combining molecular, electrophysiological, and biochemical approaches to solve some of the most basic issues in secretory biology. Our goal is to understand neurotransmitter secretion at every level and bring an integrative mind to the system we have chosen. We have many years of experience employing high resolution single vesicle amperometric and capacitance techniques, in combination with introduction of various reagents into the cell via the patch pipette to disrupt or enhance the activity of specific proteins thought to be involved in the secretory cycle. My group has made several seminal observations on the “fight-or-flight” response, the well-known reaction we all feel in situations of stress or danger. In this response the bloodstream is flooded with catecholamines, small hormones of which the best known is adrenaline (or epinephrine, familiar to any regular viewer of “ER” as it is frequently given to rev up the heart in trauma situations). The source of much of the hormone is a small gland that sits atop the kidney called the adrenal gland, the subject of our research. When stimulated during stress the gland pours adrenaline directly into the blood which then carries the hormone to various tissues, including the aforementioned heart, but also the skin (your hair stands on end), your blood vessels (relaxing them so blood flows more freely) and sweat glands (you begin to sweat profusely). Adrenaline also affects the brain, making us more alert. To understand how adrenaline is released so quickly in these situations, my laboratory studies the individual cells that make up the gland’s medulla (called chromaffin cells). To find out what is going on we use very sophisticated electrophysiological techniques that allow instantaneous (“real time”) monitoring of many parameters in these cells, including how much hormone is released (with sensitivity to resolve “single vesicle” secretion). It turns out that adrenaline is released in little “packets” due to the fact that in the gland it is stored in tiny pouches called “dense-core vesicles”. Each cell contains about 30, 000 such packets, but only a small number can be released at each time. Fusion of the vesicle with the surface of the chromaffin cell causes the hormone to be secreted into the blood. My laboratory is studying the protein molecules that allow this process to occur very efficiently. With these proteins in the right place secretion is very rapid (within 3 thousandsth of a second) but when any one of them is interfered with, secretion becomes much slower and can stop altogether. Most of our routine work is in cells from calves, as these are readily available at any slaughterhouse, but we also study human cells when available (usually from kidney donors, as the adrenal gland is not transplanted). We made the surprising observation that secretion in adult cows was significantly less than that in calves, due to the fact that an important protein (called “facilitation” L-type calcium channel) appeared to be missing. Investigating this same issue in human cells led to the same conclusion – secretion seemed much less in cells from older donors. This could be why, as many of us realize as we get older, our reactions to stressful situations take longer to develop. It might explain why, the older we are, we experience more difficulty getting out of the way of that wayward skateboarder on the sidewalk! It had been known for some time that adrenaline levels in the blood decline with age, but we might have put our finger on one of the main reasons. The consequences of such a decline might be decreased alertness and an increased delay in the “fight-or-flight” response. Our working hypothesis is that the calcium channel complement of adrenal chromaffin cells changes during development and this alters their ability to secrete rapidly, thus accounting for the reduced ability of stressful stimuli to raise plasma epinephrine in older subjects. We speculate that the decline might have an evolutionary basis – younger animals of reproductive age need to escape predators to maximize the chances of mating, while older animals that have (presumably) already reproduced are more “expendable”. Our group has made several other creative contributions to the biology of the exocytotic arm of the secretory process as it applies to dense core vesicles. While we know quite a bit about synaptic vesicle exocytosis much less attention has been paid to vesicles that release catecholamines, serotonin and virtually all peptides found in the nervous system. As it is thought that different principles might well apply to such vesicles it is important to investigate their kinetic and molecular properties. My laboratory has been instrumental in altering the current dogma about dense core vesicle secretory kinetics by using the sophisticated amperometric technique for detection of extracellular catecholamines. Previous kinetic estimates of dense core vesicle secretion had characterized it as a slow, un-localized process but with the new technology it became apparent that catecholamine release could occur in much the same time-frame as synaptic vesicle release. Using similar methodology, we turned our attention to the “quantal” nature of catecholamine release from dense-core vesicles. We have found that graded release can be observed from these vesicles under various stimulation conditions, which is quite different from the “all-or-none” dogma prevalent regarding synaptic vesicle exocytosis. Our current interest is in precisely how adrenaline gets out of the cell. We thinks that a minute passageway forms between the dense-core vesicle and the cell surface as a conduit for hormone release. In a sense the packet then “squirts” the hormone out of the cell rather than dumping its entire contents into the blood. This would be a very efficient process because the packet could then be recaptured wholesale by the cell and refilled with adrenaline, instead of having to be remade by the cell. My laboratory studies this “recycling” process (endocytosis arm of the secretory process) because we believe that very similar processes are going on in every nerve cell in the body. Indeed, the chromaffin cells are really modified nerve cells with the specialized function of secreting adrenaline instead of communicating with other nerve cells in a network. After cells secrete they must internalize the membrane rapidly in order to maintain a steady-state surface area. The actual mechanism of retrieval is obscure but there is accumulating evidence that it involves recapture of the empty intact vesicle. Moreover, the internalized vesicles are recycled rapidly back to the releasable pool at least in the nervous system, thus the mechanism is critical to maintain ongoing secretory activity.  (SLIDE 1) Neurons and neuroendocrine cells release neurotransmitters and hormones by exocytosis followed by recovery of vesicular membrane by endocytosis. The recovery of vesicular membrane at nerve terminals represent the first step in the rapid recycling of synaptic vesicles. The first observations of this type were made in the early 70's when it was realized that the total number of quanta that could be released from the frog NMJ was much greater than the total number of SVs present at that terminal. Those findings gave rise to the notion of SV recycling and the major question became how does this happen?  (SLIDE 2) Two competing theories were proposed. First, Heuser and Reese proposed that vesicles fuse by completely flattening and merging into the plasma membrane and that recovery of vesicular components for recycling is accomplished by clathrin-coated vesicles, which bud from the plasma membrane at distant sites from the sites of exocytosis. These vesicles then go on to fuse with each other to form an endosomal structure that somehow budded new synaptic vesicles. The other hypothesis was proposed by Bruno Ceccarelli, and later came to be called "kiss-and-run" exocytosis. In this mechanism, SVs fuse transiently with the presynaptic membrane, release their contents and are immediately retrieved intact directly at the active zone and may then be refilled with transmitter without the necessity of the sorting processes thought to be integral to the coated vesicle mechanism. An obvious advantage of a KR mechanism is the ability to rapidly recycle vesicles, which is an essential feature of neuronal communication, because without this process, neuronal firing would rapidly deplete the pool of transmitter-containing vesicles ready for release. Ceccarelli argued that coated vesicles only appear in nerve terminals that are subjected to large stimuli but are never seen under mild stimulation conditions. However, he lacked morphological markers for the KR pathway, which he claimed was the major mechanism of recycling under physiological conditions. Ceccarelli had the biggest disadvantage for a morphologist in that he could not point to his retrieved vesicles in electronmicrographs as they would have exactly the same appearance as SVs. Heuser and Reese on the other hand based their model on the few CCVs they observed in their micrographs and that is now repeated in all the Neuroscience textbooks.  (SLIDE 3) Although FCF mode of secretion is still the prevailing dogma, strong new evidence supporting “kiss-and-run” mechanisms in neurons and neuroendocrine cells has emerged over the last two decades  (SLIDE 4) The first body of evidence supporting the “kiss-and-run” mode of secretion has been obtained with the use of fluorescent dyes such as FM1-43. This dye which is internalized during endocytosis from the plasma membrane can be released from the cells within 30 sec. Thus the whole recycling pathway may take place in well under 1 min. This immediately raises questions for the “coated vesicle hypothesis”. Although the KR fusion process has been suggested to occur at several types of synapses, the results implicating or eliminating the KR fusion process are controversial and inconclusive because the techniques used do not provide direct evidence for or against the KR mechanism.  (SLIDE 5) The second body of evidence that supports the “kiss and run” mode of secretion is the discovery of “rapid endocytosis” by capacitance measurements in a number of secretory cells including neurons.  (SLIDE 6) In 1993 I initiated the study of “rapid endocytosis” in adrenal chromaffin cells, a highly characterized cell model for neurotransmitter release. These cells secrete a variety of products, of which catecholamines are the most important, directly into the bloodstream, and this is responsible for the rapid burst of serum catecholamines that appears under stressful conditions, otherwise known as "adrenaline rush".  (SLIDE 7) To directly assess endocytosis I used capacitance measurements of cell surface area to estimate both exo and endocytosis in a single stroke. When AC cells are stimulated by short trains of depolarizing pulses or APs, the cells secrete as reflected in the upward steps in the Cm trace. Under these circumstances Cm increases in a series of steps reflective of fusion of DCVs with the plasma membrane. For the first time, we reported the existence of two different endocytotic processes and their dependence on stimulation intensity.  (SLIDE 8) Shown here is a continuous Cm recording from a cell sequentially stimulated with two protocols designed to elicit rapid endocytosis or slow endocytosis. When cells are subjected to brief stimulation, Cm rises (exocytosis) but then rapidly declines to baseline within ~20 sec, which I described as rapid endocytosis. However, when stimulation is increased several-fold, rapid endocytosis disappears by the end of the stimulation period. Instead, a slower form of membrane retrieval ensues, which gradually returns Cm to baseline within ~10 min, which we described as slow endocytosis. For the first time, we verified that these two modes of membrane recovery are kinetically and mechanistically divergent.  (SLIDE 9) Rapid endocytosis is a Ca2+-dependent process, replacement of extracellular Ca2+ with either Ba2+ or Sr2+ ablates the process while allowing exocytosis to continue.  (SLIDE 10) Calmodulin is the Ca2+-receptor for rapid endocytosis. Anti-CaM monoclonal antibodies completely eliminated rapid endocytosis without affecting secretion or Ca currents  (SLIDE 11) Affinity-purified anti-clathrin heavy chain monoclonal antibodies had no effect on rapid endocytosis. Rapid endocytosis requires intact microtubules as it is blocked by nocodazole treatment, but it is independent of acidification of the cytosol. Therefore, rapid endocytosis, is elicited by mild to moderate stimulation, is a process with a half time of msec-sec, is Ca2+-, calmodulin-, dynamin-1, and microtubule-dependent, but is a clathrin-independent process.  (SLIDE 12) Shown here are Cm records from cells in which secretion was evoked for both rapid endocytosis and slow endocytosis. With a pipette solution containing 120 mM K+, slow endocytosis takes place and is completed within ~ 8 min. However, removal of K+ had no effect on rapid endocytosis, but completely abolished slow endocytosis.  (SLIDE 13) To examine whether slow endocytosis depends on the assembly of clathrin cages, I introduced into the cell affinity-purified monoclonal anti-clathrin antibody, that does not influence rapid endocytosis, but effectively blocked slow endocytosis. To examine the Ca2+ dependence of slow endocytosis, I replaced extracellular Ca2+ with either Ba2+ or Sr2+, (rapid endocytosis is abolished under these conditions), but slow endocytosis still occurs, suggesting that it is independent of Ca2+ entry, or has a divalent cation specificity different to rapid endocytosis.  (SLIDE 14) As confirmed in the immunoblots shown on the next two slides, AC cells express both dynamin-1 and dynamin-2. To establish whether rapid endocytosis and slow endocytosis depend on the function of different dynamin isoforms, I diffused anti-dynamin-1 and anti-dynamin-2-specific antibodies into the cells before stimulation. Shown here are Cm records from cells in which affinity-purified anti-dynamin-1-specific IgG was introduced into the cell followed by transient or sustained stimulation. Anti-dynamin-1 IgG inhibits rapid endocytosis but has no effect on slow endocytosis.  (SLIDE 15) By contrast, anti-dynamin-2 IgG inhibits slow endocytosis but has no effect on rapid endocytosis. These results demonstrate for the first time that different dynamin isoforms are preferentially used by the two processes: dynamin-1 mediates rapid endocytosis while dynamin-2 mediates slow endocytosis.  (SLIDE 16) More recently, we found that antibodies to amphiphysin inhibited slow endocytosis, but lacked effect on rapid endocytosis. Our results suggested that amphiphysins are exclusively associated with clathrin- and dynamin-2-dependent slow endocytosis. Therefore, slow endocytosis, is activated with sustained stimulation, is completed within 10 min, involves clathrin, dynamin-2, and has a divalent cation specificity different to rapid endocytosis. These results are the first demonstration that AC cells exhibit two kinetically and mechanistically distinct forms of endocytosis that are coupled to different extent of exocytosis and are mediated by different dynamin isoforms.  (SLIDE 17) Based on these data, we surmised that RE is associated with “kiss-and-run” mechanism of transmitter release, and that it is the prevalent means of vesicle recapture and recycling under normal physiological conditions, while the clathrin-based SE only comes into play at higher levels of stimulation and may be associated with “full-collapse-fusion” of vesicles with the plasma membrane. Because of the collapse, DCV proteins must now be sorted from plasma membrane proteins and this in part might be accomplished by CCVs.  (SLIDE 18). The third body of evidence that supports the “kiss and run” mode of secretion is the demonstration that partial release of catecholamines may be the rule under physiological conditions.  (SLIDE 19) We determined the characteristics of individual secretory events by measuring catecholamine release by amperometry using an extracellular carbon fiber electrode gently apposed to the cell surface, while we stimulated the cells with trains of APs. To assess the effect of the pattern of stimulation on the kinetics of secretion, we modified (1) the stimulation frequency, or (2) the external [Ca2+]o. Analysis of individual amperometric spikes demonstrated that single secretory events exhibit significant variation depending on stimulation conditions. At low frequencies or low [Ca2+]o, only very small amplitude spikes were recorded, but increasing the frequency to 1Hz or the [Ca2+]o to 0.75 mM led to the appearance of a population of spikes that were larger and wider. Finally, at 7Hz and 2 mM [Ca2+]o, most spikes were large and narrow. These results represent the first systematic test of the quantal hypothesis and suggest that DCVs may release only part of their contents during each round of secretion, allowing the speculation that fractional release of catecholamines may be the rule under normal physiological conditions.  (SLIDE 20). Then, we hypothesized that the partial release may be linked to the KR mechanism of neurotransmitter release. To test it, we investigated the possibility that RE is directly linked to fusion pore closure and therefore would play a role in quantal size modulation. We found that during successive stimulation periods, intracellular application of anti-dynamin antibodies, progressively reduced the total amount of catecholamine released and modified the kinetics of single secretory events. The extent of catecholamine secretion was reduced by a maximum of 76% at the fourth round of stimulation, suggesting a substantial attrition of the release-ready vesicle pool when RE is blocked. At 2 mM [Ca2+]o, the amperometric recordings revealed that anti-dynamin IgGs caused dramatic spike broadening in a subset of single secretory events. These broad spikes represent only ~30% of the ASs remaining, while the other ~70% comprised small spikes, and resembled those seen in low [Ca2+]o conditions. Indeed, inhibition of RE had no effect on the extent of secretion, when stimulation was carried out in low [Ca2+]o. These results indicate that the efficiency of RE modulates the amount of transmitter released, possibly by altering the kinetics of fusion pore closure, and that RE itself plays an essential role in the recycling of vesicles needed for continuous secretion.  (SLIDE 21). In light of the evidence for subquantal release, we proposed this model:(Step 1) At low level of stimulation, when the Ca2+ signal is small, the fusion pore transiently opens and closes, allowing only a small amount of catecholamines to be released (detected as small ASs). (Steps 2-3) With increased stimulation, [Ca2+]i rises, fusion pore dilatation is enhanced, allowing more catecholamine to be release but quite inefficiently, as seen in the types of ASs observed. (Step 4) More stimulation raises [Ca2+]i to a threshold that triggers dilatation of the fusion pore to its maximal conductance, allowing fast and efficient release of catecholamines. However, elevated [Ca2+]i also hastens fusion pore closure, thus restricting the amount of hormone released, which is reflected in large but narrow ASs that predominate under such conditions. (Step 6) When RE is blocked by anti-dynamin IgGs, larger amplitude ASs exhibit prolonged duration, allowing more catecholamine to be released (seen as a 3.3-fold increase of the average charge of the individual AS). Therefore, under conditions of abrogated RE or with tetanic stimulation, degranulation can occur with complete incorporation of the DCV membrane into the plasma membrane, followed by slow endocytosis, mediated by clathrin-coated vesicles.  (SLIDE 22). The fourth body of evidence that supports the “kiss and run” mode of secretion has been obtained with the use of cell-attached capacitance technique to record single vesicle fusion events. The capacitance recording technique combined with the analysis of fusion pore conductance provides the most convincing direct evidence for the KR mechanism, which is proposed to occur via a fusion pore connecting transiently the vesicle to the plasma membrane. Therefore, cell-attached capacitance is the only direct approach to unravel the process of fusion of single vesicles with the plasma membrane. However, although cell-attached capacitance is the first direct approach to demonstrate the existence of KR and FCF modes of secretion, none of the above studies measured the fusion pore under relevant physiological conditions. Those observations were based solely on spontaneous release or with non-physiological maneuvers.  (SLIDE 23). The fifth body of evidence that supports the “kiss and run” mode of secretion has been obtained with the use of “double patch-clamp” approach to record single vesicle fusion under physiological condition. To date, the lack of direct measurement of fusion pore kinetics under physiological conditions has hindered an evaluation of the prevalence and physiological relevance of the KR mode of secretion. However, my laboratory using a “double patch-clamp” approach, has reported that “kiss-and-run” is the predominant mode of secretion under moderate stimulation conditions, that [Ca2+]i controls the transition between KR and FCF, each of which is coupled to different modes of endocytosis .  (SLIDE 24). This is a diagram showing the “double patch-clamp” approach in two different configurations: (A) cell-attached combined to whole-cell recordings; or (B) amperometry combined to whole-cell recording. The cell-attached configuration records single vesicle fusion events and fusion pore properties. The carbon fiber electrode detects catecholamine released from single vesicles. The whole-cell configuration, is used to stimulate the cell with APs; or dialyze the cell with the Ca2+ indicator aequorin; or with immunological and molecular probes, etc  (SLIDE 25) In this experiment, we stimulated cells with the nicotinic agonist DMPP (10 μM) and recorded in cell-attached the resulting single vesicle fusion events. Here is shown sequential capacitance up- and down-steps of the same size, which reflects fusion of single vesicles with the surface membrane. These records reveal that, at a small membrane-patch, single vesicles undergo fusion and retrieval rapidly with a measurable fusion pore conductance. It was immediately apparent that the majority of the single vesicle fusion events represented “kiss-and-run”. In this experiment, (169) “kiss-and-run” events were observed, but only (18) FCF events were seen. Each transient event is characterized by measuring: (1) vesicle capacitance (Cv), (2) fusion pore conductance (Gp), and (3) the event duration.  (SLIDE 26). In this experiment, using the “double-patch-clamp” approach we recorded single fusion events is response to stimulation of the cell with APs. We found that APs at 1 Hz or 7Hz trigger single exocytotic events almost entirely by a “kiss-and-run” mechanism. In this experiment, APs at 1Hz triggered (69) “kiss-and-run” events, with no FCF events that were apparent at this frequency. APs at 7Hz triggered more FCF events (14), but “kiss-and-run” events still predominated (150). Cumulative data from “kiss-and-run” events are shown as histograms. APs at 7 Hz reduced the mean unitary event duration significantly from 107 to 76 msec, and increased the mean value of Gp for a subset of secretory events (290 pS). These results suggest that higher [Ca2+]i, consequently after an increase in stimulation frequency, increases the fusion pore conductance and speeds up its closure, as well as promotes FCF events. To test these hypotheses, we stimulated cells in the presence of different levels of free [Ca2+]i.  (SLIDE 27). In this experiment, different levels of free [Ca2+]i were delivered to the cell interior through the whole-cell pipette; and exocytosis was monitored concurrently recording both whole-cell and cell-attached capacitance. At 10 μM [Ca2+]i, unitary fusion events in the cell-attached patch were predominantly “kiss-and-run” events (B). When free [Ca2+]i was increased to 102 μM, both “kiss-and-run” and FCF events were seen, and sometimes both types of events were recorded in the same patch (C). In contrast, predominantly FCF events were detected when free [Ca2+]i was increased to 210 μM. (E) The occurrence probability of secretory events shows a shift toward FCF at the expense of “kiss-and-run” events at a higher level of [Ca2+]i. The numbers in parenthesis are the number of events in each condition. These results suggest that prevailing [Ca2+]i controls the transition between “kiss-and-run” and “FCF”. It is possible that the principal effect of [Ca2+]i is on fusion pore closure, which in turn might be related to the mechanism of endocytosis. Our finding that “kiss-and-run” events were virtually absent at 210 μM raised the possibility that RE is mechanistically associated with “kiss-and-run” and FCF becomes the norm when RE is abrogated. To further test this possibility, we performed the following experiment.  (SLIDE 28) Previously, I showed that brief and sustained physiological stimulations trigger two modes of endocytosis, RE and SE, respectively. I have also shown that replacement of extracellular Ca2+ with either Ba2+ or Sr2+did not affect SE significantly, but completely inhibited RE. In these experiments, both of these paradigms were used to investigate the connection between RE and “kiss-and-run” fusion events. Brief stimulation caused reproducible RE in the whole-cell configuration (Bb1) and only KR fusion events in the cell-attached patch (Bb2). In contrast, when Sr2+ replaced bath Ca2+, the brief stimulation resulted in only exocytosis with RE being completely blocked (Cc1). At the same time in the cell-attached patch, only FCF events were recorded (Cc2). Three FCF events were recorded in the cell-attached trace. The conductance trace (Re) shows a brief transient before the fusion pore expands fully. Blockade of RE does not affect fusing vesicle size. Furthermore, when more sustained stimulation was used in the presence of Ca2+ as charge carrier, the robust Cm increase was followed by a slow decrease of Cm (SE) that takes ~11 min to return to baseline value (Dd1). Note that RE does not take place as shown in the whole-cell recording, whereas in the cell-attached patch, only FCF events were apparent (Dd2). Concurrently, two FCF events were evoked in the cell-attached capacitance recording. These results suggest that, at moderate physiological stimulations, RE is intimately coupled to “kiss-and-run” fusion type of secretion, whereas FCF events are followed by SE at more sustained stimulations.  (SLIDE 29). Considering these data we propose this model to illustrate the modulation of the fusion pore by [Ca2+]i and the role it plays in exocytosis and vesicle recycling. Different [Ca2+]i governed by different patterns of physiological stimulation give rise to three alternative, kinetically and mechanistically distinct modes of fusion, and each of which is coupled to different modes of vesicle recycling. (Mode 1) Al low-stimulation frequency (low [Ca2+]i), transient fusion is the major mechanism of vesicle exocytosis, in the mode of “kiss-and-stay”. Vesicles fuse with the plasma membrane for a longer duration (~107 ms) through a narrow fusion pore (~150 pS). Therefore, a small amount of transmitter is released for a longer duration. (in line with the amperometry data showing ASs small and wide). These spikes were not affected by antagonism of dynamin function, suggesting that closure of the fusion pore at this level of [Ca2+]i may simply be a reversal of the fusion reaction. In these conditions, the vesicle could undergo few rounds of release with no need to undock and refill with transmitters. This mode of release is important to maintain a low basal level of release, especially if the release ready pool contains a small number of vesicles. (Mode 2) At moderate stimulation (medium [Ca2+]i), transient fusion is again the major mechanism of vesicle exocytosis, in the mode of “kiss-and-run”. Vesicles fuse transiently but for a shorter duration (~70 ms) and higher pore conductance (~250 pS). Secretion is very efficient, (in line with the amperometry data showing large but narrow ASs), and the same vesicle is retrieved intact and could be reused, each time with partial quantal release. Because RE involves Ca2+ and dynamin-1, it is most likely that this level of [Ca2+]i corresponds to a specific threshold, which could trigger the formation of a dynamin-1 ring around the neck of the retracting vesicle, facilitating the closure of the fusion pore and initiating fission. (Mode 3) Under sustained stimulation (high [Ca2+]i), vesicles will dump their entire content into the bloodstream (in line with the amperometry data showing large and very wide ASs; seen as a 3.3-fold increase of the average charge of the individual AS) and the fusion pore expands irreversibly promoting FCF with the surface membrane. At sustained stimulation (high [Ca2+]i), dynamin-1-dependent RE is blocked. Therefore, absence of dynamin-1 rings around the neck of the vesicle might allow the fusion pore, which is already at high conductance in presence of high [Ca2+]i, to expand until full-collapse-fusion of the vesicle with the plasma membrane. These vesicles would be retrieved by the clathrin- and dynamin-2-dependent SE and recycle through the endosomal compartment. High [Ca2+]i favors the incidence of FCF while preserving the KR to conserve vesicular resources. Our preliminary data indicates that high [Ca2+]i is not homogeneous allowing some spots of medium [Ca2+]i, preserving KR at release sites even at sustained stimulation.  (SLIDE 30). The data I have presented establish KR and RE as the dominant mode of exocytosis and endocytosis respectively, and identify the frequency of stimulation as a factor that modulates their prevalence and physiological role. These studies suggest that transient fusion and reuse of vesicles serves to conserve resources during periods of low-moderate frequency firing, and thereby ensures that neurosecretory cells are ready to respond to the demands of high-frequency with a full quota of fusion-competent vesicles. The ongoing work in my laboratory aims to define further the precise conditions under which transient fusion and FCF predominate, and how each contributes to maintaining neurosecretion over the wide range of physiological frequency that cells experience in vivo.  (SLIDE 31)  (SLIDE 32) Selected Publications with available hyperlinks:
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