Electron microscopic autoradiography of serotonin uptake in the ganglia of the fresh-water mussel (Anodonta cygneak L.).

After in vitro incubation, the uptake of labelled serotonin was investigated by electron microscopic autoradiography in the ganglia of fresh-water mussel (Anodonta cygnea L.) The labelled serotonin was primarily taken up by the axons of the neurons. The silver grains could always be localized over axons containing eccentric dense-core vesicles with a diameter of 100--200 nm. The results suggest (a) the possibility of the electron microscopic identification of serotonin-containing neurons, and (b) the direct role of the eccentric dense-core vesicles in the storage of serotonin.     

Dense-core vesicles and non-synaptic exocytosis in the central body of the crayfish brain

Summary The central body in the median protocerebrum of the brain of the crayfish Cherax destructor is a distinctive area of dense neuropile, the nerve fibres of which contain three main types of vesicles: electronlucent vesicles (diameter 35 nm), dense-core vesicles (diameter 64 nm), and large structured dense-core vesicles (diameter 98 nm, maximum 170 nm). Different vesicle types were found together in the same neurons. Electronlucent vesicles were seen at presynaptic sites and rarely observed in the state of exocytosis. Exocytosis of densecore and structured dense-core vesicles was a regular feature on non-synaptic release sites either close to, or at some distance from pre- and subsynaptic sites. Non-synaptic exocytotic sites are more often observed than chemical synapses. Different forms of exocytosis seen at non-synaptic sites included the release of single densecore vesicles, packets of dense-core vesicles, and rows of dense-core vesicles lined up along cell membranes and around fibre invaginations. Swelling and the enhanced electron density of extracellular non-synaptic spaces may mark the positions of prior exocytotic events. In vitro treatment of the brain with tannic acid buffer solution followed by conventional double fixation resulted in the augmentation of non-synaptic exocytosis. Electron microscopy of proctolin- and serotonin-immunoreactive nerve fibres shows them to contain dense-core and electron-lucent vesicles and to be surrounded by many unlabelled profiles similarly laden with dense-core vesicles and electron-lucent vesicles, indicating the presence of other, not yet identified, neuroactive compounds.     

Subcellular localization of chromogranins, calcium channels, amine carriers, and proteins of the exocytotic machinery in bovine splenic nerve

Subcellular fractionation of bovine splenic nerves, which consist mainly of sympathetic nerve fibers, has been useful for characterizing cellular organelles en route to the terminal. In the present study we have characterized the subcellular distribution of both secretory and membrane proteins. A newly discovered chromogranin-like protein, NESP55, was found in large dense-core vesicles. The endogenous processing of NESP55 was comparable to that of chromogranins but more limited than that of secretogranin II and chromogranin B. For membrane proteins three major types of distribution were found. The amine carrier VMAT2 was confined to large dense-core vesicles. VAMP or synaptobrevin was present both in large dense-core vesicles and in lighter vesicles, whereas SNAP-25, syntaxin, and two types (N and L) of Ca2+ channels were found in a special population of lighter vesicles but were not present in large dense-core vesicles or at the most in very low concentrations. The plasma membrane norepinephrine transporter was apparently present in a separate type of vesicle, but this requires further study. These results further characterize vesicles en route to the terminal and establish for the first time that peptides involved in exocytosis (syntaxin, SNAP-25, and N- and L-type Ca2+ channels) are apparently transported to the terminal in a special type of vesicle. The exclusive presence of the amine carrier in large dense-core vesicles indicates that the formation of small dense-core vesicles in the terminals requires a reuse of membrane components of large dense-core vesicles.     

Ultrastructural aspects of the paraventricular nuclei in the rat

Summary The ultrastructural aspects of the paraventricular nucleus and its neuropil are described in the normal rat.Two types of neurons can be distinguished morphologically. The first type contains numerous dense-core vesicles (mean diameter: 140 nm). The cisternae of the endoplasmic reticulum are arranged parallely at the periphery of the cell body.The second type of neuron contains a few dense-core vesicles (mean diameter: 75 nm) and the endoplasmic reticulum is randomly distributed in the cytoplasm. In the neuropil, two types of dense-core vesicles are observed in separated axons. The histogram of the distribution of their mean diameter clearly indicates a double population of vesicles.The signification of the second type of neuron in the paraventricular nucleus is discussed and its possible relation to TRF synthesis is evoked.This work was supported by a grant from the Belgian National Fund for Scientific Research.The author wish to thank Mrs. Hunninck-Couck for her devoted and skillful technical assistance.     

Ultrastructural organization of the anterior sensory neuropile in the metathoracic ganglion of the locust Locusta migratoria]

The study of serial sections of the metathoracic ganglion of Locusta migratoria showed that the fibres of the tympanal nerve terminate in the dorsal half of the anterior sensory neuropile (ASN). Three types of synaptic endings were found in the ASN. Endings type I contain dense-core vesicles 600-850 A in diameter, more or less uniformly distributed in the axoplasm. They do not form specialized contacts with postsynaptic fibres and are localized only in the most ventral part of the ASN. Endings type II contain clear round vesicles 400-450 A in diameter (rare 250-300 A) and form typical synapses with dense pre-and postsynaptic membranes and synaptic cleft 150-200 A. Four types of contacts formed by these endings with postsynaptic fibres were found: 1 : 1 synapses; convergent, divergent and serial. All of them are well presented in the auditory neuropile. Endings type III contain both dense-core and clear vesicles in different relation. Only clear vesicles of these endings are connected with the active sites of the membrane.     

Synapses in the rat substantia nigra

The composition and organization of the input to the rat substantia nigra were studied with the electron microscope. Four distinct types of synaptic boutons were described. The first contained small (381 A), clear synaptic vesicles. The second type contained the small, clear vesicles and several large, dense-core vesicles. The third ending contained large, dense-core vesicles and larger (581 A) clear vesicles. The fourth ending, found on the axon hillock and other terminal boutons, contained slightly elongated, clear synaptic vesicles. The presence of these four boutons was discussed in light of the known afferent input and neurochemical composition of the substantia nigra.     

Synaptology of neurosecretory cells in the nucleus paraventricularis of the domestic fowl

Summary Processes of magnocellular neurosecretory cells (MNCs) are easily identifiable on the basis of their content in neurosecretory granules in the neuropil of the rostral division of the paraventricular nucleus (PVN) of the domestic fowl. In specimens sacrificed during the winter the synaptic organization of the neuropil and the pattern of synapses ending on neurosecretory processes were studied at the ultrastructural level. Synapses in the rostral part of the PVN neuropil may be divided into three main categories on the basis of their morphology and their content of clear and dense-core synaptic vesicles. These different types of terminals can be attributed to aminergic, peptidergic or other types of synapses. The percent distribution of synapses within these categories differs when all synapses observed in the neuropil or only those ending on MNC processes are compared. Present ultrastructural data obtained in birds support two physiological hypotheses already suggested for mammals, i.e., the probable existence of a recurrent pathway to MNCs via an interneuron, and the importance of aminergic and peptidergic input in regulating the electrical activity of MNCs.This work was partly supported by a CNR grant (n. 81.00377.04)     

Nerve growth factor-dependent sorting of synaptotagmin IV protein to mature dense-core vesicles that undergo calcium-dependent exocytosis in PC12 cells

Synaptotagmin IV (Syt IV) is a fourth member of the Syt family and has been shown to regulate some forms of memory and learning by analysis of Syt IV null mutant mice (Ferguson, G. D., Anagnostaras, S. G., Silva, A. J., and Herschman, H. R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5598-5603). However, the involvement of Syt IV protein in vesicular trafficking and even its localization in secretory vesicles are still matters of controversy. Here we present several lines of evidence showing that the Syt IV protein in PC12 cells is normally localized in the Golgi or immature vesicles at the cell periphery and is sorted to fusion-competent mature dense-core vesicles in response to short nerve growth factor (NGF) stimulation. (i) In undifferentiated PC12 cells, Syt IV protein is mainly localized in the Golgi and small amounts are also present at the cell periphery, but according to the results of an immunocytochemical analysis, they do not colocalize with conventional secretory vesicle markers (Syt I, Syt IX, Rab3A, Rab27A, vesicle-associated membrane protein 2, and synaptophysin) at all. By contrast, limited colocalization of Syt IV protein with dense-core vesicle markers is found in the distal parts of the neurites of NGF-differentiated PC12 cells. (ii) Immunoelectron microscopy with highly specific anti-Syt IV antibody revealed that the Syt IV protein in undifferentiated PC12 cells is mainly present on the Golgi membranes and immature secretory vesicles, whereas after NGF stimulation Syt IV protein is also present on the mature dense-core vesicles. (iii) An N-terminal antibody-uptake experiment indicated that Syt IV-containing vesicles in the neurites of NGF-differentiated PC12 cells undergo Ca(2+)-dependent exocytosis, whereas no uptake of the anti-Syt IV-N antibody was observed in undifferentiated PC12 cells. Our results suggest that Syt IV is a stimulus (e.g. NGF)-dependent regulator for exocytosis of dense-core vesicles.     

Hypophysiotrophic thyrotropin releasing hormone (TRH) synthesizing neurons. Ultrastructure, adrenergic innervation and putative transmitter action

The neuropeptide thyrotropin releasing hormone (TRH) is capable of influencing both neuronal mechanisms in the brain and the activity of the pituitary-thyroid endocrine axis. By the use of immunocytochemical techniques, first the ultrastructural features of TRH-immunoreactive (IR) perikarya and neuronal processes were studied, and then the relationship between TRH-IR neuronal elements and dopamine-beta-hydroxylase (DBH) or phenylethanolamine-N-methyltransferase (PNMT)-IR catecholaminergic axons was analyzed in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN). In control animals, only TRH-IR axons were detected and some of them seemed to follow the contour of immunonegative neurons. Colchicine treatment resulted in the appearance of TRH-IR material in parvocellular neurons of the PVN. At the ultrastructural level, immunolabel was associated with rough endoplasmic reticulum, free ribosomes and neurosecretory granules. Non-labelled axons formed synaptic specializations with both dendrites and perikarya of the TRH-synthesizing neurons. TRH-IR axons located in the parvocellular units of the PVN exhibited numerous intensely labelled dense-core and fewer small electron lucent vesicles. These axons were frequently observed to terminate on parvocellular neurons, forming both bouton- and en passant-type connections. The simultaneous light microscopic localization of DBH or PNMT-IR axons and TRH-synthesizing neurons demonstrated that catecholaminergic fibers established contacts with the dendrites and cell bodies of TRH-IR neurons. Ultrastructural analysis revealed the formation of asymmetric axo-somatic and axo-dendritic synaptic specializations between PNMT-immunopositive, adrenergic axons and TRH-IR neurons in the periventricular and medial parvocellular subnuclei of the PVN. These morphological data indicate that the hypophysiotrophic, thyrotropin releasing hormone synthesizing neurons of the PVN are directly influenced by the central epinephrine system and that TRH may act as a neurotransmitter or neuromodulator upon other paraventricular neurons.     

Caudal neurosecretory system of the zebrafish: ultrastructural organization and immunocytochemical detection of urotensins

The caudal neurosecretory system is described here for the first time in the zebrafish, one of the most important models used to study biological processes. Light- and electron-microscopical approaches have been employed to describe the structural organization of Dahlgren cells and the urophysis, together with the immunohistochemical localization of urotensin I and II (UI and UII) peptides. Two latero-ventral bands of neuronal perikarya in the caudal spinal cord project axons to the urophysis. The largest secretory neurons (~20 μm) are located rostrally. UII-immunoreactive perikarya are much more numerous than those immunoreactive for UI. A few neurons are immunopositive for both peptides. Axons contain 75-nm to 180-nm dense-core vesicles comprising two populations distributed in two axonal types (A and B). Large dense vesicles predominate in type A axons and smaller ones in type B. Immunogold double-labelling has revealed that some fibres contain both UI and UII, sometimes even within the same neurosecretory granule. UII is apparently the major peptide present and predominates in type A axons, with UI predominating in type B. A surprising finding, not previously reported in other fish, is the presence of dense-core vesicles, similar to those in neurons, in astrocytes including their end-feet around capillaries. Secretory type vesicles are also evident in ependymocytes and cerebrospinal-fluid-contacting neurons in the terminal spinal cord. Thus, in addition to the urophysis, this region may possess further secretory systems whose products and associated targets remain to be established. These results provide the basis for further experimental, genetic and developmental studies of the urophysial system in the zebrafish.     

The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott-Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.     

On catecholamine-containing cells in the rat interatrial septum

Summary The interatrial septum of the rat heart contains cells which show a strong intensive-yellow paraformaldehyde-induced fluorescence. By electron microscopy these cells are characterized by an abundance of dense-core vesicles.Cholinergio axons form axo-somatic synaptic contacts with the catecholamine-containing cells. These cells, packed with dense-core vesicles, are frequently interdigitated and interconnected by zonulae and maculae adhaerentes and occludentes. The catecholamine-containing cells are surrounded by satellite cells either individually or in groups.The catecholamine-containing cells, which bear blunt, plumpish processes, can be subdivided, on the basis of position and morphology into two types. One class of cells lies within the fibroblast capsule of the intra-atrial ganglion (van der Zypen, Hasselhorst, Merz and Fillinger, 1974). A second aggregation of catecholamine-containing cells occurs outside the ganglia in close proximity to capillaries. The capillaries exhibit pores in the area of contact with the catecholaminergic cells. The structure of these catecholamine-containing cells is described and their possible function discussed.

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Zusammenfassung Im Septum interatriale des Rattenherzens treten Zellen in Erscheinung, die nach Paraformaldehyd-Bedampfung eine intensive hellgelbliche Fluoreszenz zeigen. Diese Zellen zeichnen sich durch einen großen Reichtum an dense-core vesicles aus. Cholinerge Axone bilden axo-somatische Synapsen an den katecholaminhaltigen Zellen aus. Die mit dense-core vesicles angefüllten Zellen sind oft ineinander verzahnt und durch Zonulae adhaerentes verbunden. Einzeln oder in Gruppen werden die katecholamin-enthaltenden Zellen von Satelliten-Zellen umgeben.Die mit kurzen plumpen Fortsätzen versehenen katecholaminhaltigen Zellen lassen aufgrund ihrer Lage und eines andersartigen Baues zwei Typen erkennen. Eine Gruppe von Zellen liegt innerhalb der Fibrozytenkapsel des Ganglion intraatriale (van der Zypen, Hasselhorst, Merz und Fillinger, 1974). Eine zweite Ansammlung von Katecholamin enthaltenden Zellen findet sich außerhalb der Ganglien in engem Kontakt zu Kapillaren. Die Kapillaren weisen im Bereich des Kontaktes mit den katecholaminergen Zellen Poren auf. Die Struktur dieser Zellen wird geschildert und ihre mögliche Funktion diskutiert.

     

Granule containing cells and fibres in the sinus venosus of elasmobranchs

The concentrations of catecholamines in the heart chambers of elasmobranchs were measured by the fluorimetric method of Bertler et al. (1958). Noradrenaline (NA) can be detected in all the chambers, but the sinus venosus is by far the richest in NA. This can either be due to the presence of storage sites for this amine in the sinus wall, or to a transport of amine to the sinus venosus from the anterior chromaffin bodies. The sinus wall contains large numbers of "granule containing cells" and axon-like processes, both with numerous dense-core vesicles of about 1800 A diameter. The dense-core vesicles contain a uranophilic matrix indicating the presence of protein, phospholipids and/or nucleic acid. The reactions failed to demonstrate amine, which may be due to a loss of amine by diffusion, to a relatively low intravesicular amine concentration, or, to the absence of amines in these granule-containing cells and processes. Heavy accumulations of granule-containing processes occur in the subendothelial area. The endothelium contains fenestrae and pores through which granule-containing fibres protrude into the venous cavity. Granule-containing cells are innervated by presumed cholinergic nerve endings. It is suggested that the granule-containing cells and fibres belong to the neurosecretory system with a cholinergic input, releasing the contents of the dense-core vesicles into the blood stream at the level of the venous cavity.     

Lymphoid cells in the Harderian gland of birds

Summary Numerous secretory parvocellular perikarya were found in the preoptic region of the domestic fowl (Gallus gallus). The dense-core secretory vesicles belong to two categories: vesicles with a diameter of (i)80–90 nm and (ii) 110–140nm. Scattered magnocellular elements display larger dense-core granules. The parvocellular neurons form unit-like clusters, showing also zones of direct apposition of neuronal membranes. The surrounding neuropil is rich in synaptic structures, formed by at least three types of axon terminals, distinguishable on the basis of vesicular morphology. These observations confirm the findings in other avian species. The hypothetical function of this system of peptidergic neurons in the rostral hypothalamus of birds is discussed.     

The polybasic sequence in the C2B domain of rabphilin is required for the vesicle docking step in PC12 cells

Rabphilin is generally thought to be involved in the regulation of secretory vesicle exocytosis in neurons and neuroendocrine cells, and it has recently been hypothesized that the C2B domain of rabphilin promotes the docking of dense-core vesicles to the plasma membrane through simultaneous interaction with a vesicle protein, Rab3A/27A, and a plasma membrane protein, SNAP-25 (synaptosome-associated protein of 25 kDa). However, the physiological significance of the rabphilin-SNAP-25 interaction in the vesicle-docking step has never been elucidated. In this study we demonstrated by a mutation analysis that the polybasic sequence (587 KKAKHKTQIKKK 598) in the C2B domain of rabphilin is required for SNAP-25 binding, and that the Asp residues in the Ca(2+)-binding loop 3 (D628 and D630) of the C2B domain are not required. We also investigated the effect of Lys-->Gln (KQ) mutations in the polybasic sequence of the C2B domain on vesicle dynamics by total internal reflection fluorescence microscopy in individual PC12 cells. A rabphilin(KQ) mutant that completely lacks SNAP-25-binding activity significantly decreased the number of plasma-membrane-docked vesicles and strongly inhibited high-KCl-induced dense-core vesicle exocytosis. These results indicate that the polybasic sequence in the C2B domain functions as an effector domain for SNAP-25 and controls the number of 'releasable' vesicles docked to the plasma membrane.     

Ultrastructural changes in granule cell somata and mossy fibers of the rat hippocampus during picrotoxin-induced convulsions

Summary Ultrastructural changes in hippocampal granule cells, mossy fibers and mossy fiber boutons were examined following the administration of picrotoxin in adult rats. Generalized seizures occurred within 5–10 min after the intraperitoneal injection of picrotoxin. The electron-microscopic examination of hippocampal tissues from rats that had been perfused with fixative during the seizure revealed that the large dense-core vesicles increased in number and accumulated on the presynaptic membranes of mossy fiber boutons; some of these vesicles appeared to be fused with the membranes, and omega-shaped exocytotic profiles were frequently seen. Furthermore, greatly increased numbers of coated vesicles (60–90 nm in diameter) were observed on the maturing faces of Golgi fields of granule cells. Thus, our study not only indicates an increased incidence of exocytosis of large dense-core vesicles during picrotoxin-induced seizures, but also suggests that these vesicles are replaced in excess from the perikaryon of the granule cell.     

Electron-microscopic investigations of vasoactive intestinal peptide (VIP)-like immunoreactive terminal formations in the lateral septum of the pigeon

Vasoactive intestinal peptide (VIP)-like immunoreactive terminal fields were examined in the lateral septum of the pigeon by means of immunocytochemistry. According to light-microscopic observations, these projections originated from VIP-like immunoreactive cerebrospinal fluid (CSF)-contacting neurons, which are located in the ependymal layer of the lateral septum and form a part of the lateral septal organ. The processes of these cells gave rise to dense terminal-like structures in the lateral septum. Pre-embedding immuno-electron microscopy revealed that VIP-like immunoreactive axon terminals had synaptoid contacts with perikarya of small VIP-immunonegative neurons of the lateral septum, which were characterized by an invaginated nucleus, numerous mitochondria, a well-developed Golgi apparatus, endoplasmic reticulum and a small number of dense-core vesicles (about 100 nm in diameter). VIP-like immunoreactive axons were also seen in contact with immunonegative dendrites in the lateral septum. In both axosomatic and axodendritic connections, VIP-like immunoreactive presynaptic terminals contained large dense-core vesicles, clusters of small vesicles and mitochondria. These findings suggest that VIP-immunoreactive neurons of the lateral septal organ project to small, presumably peptidergic nerve cells of the lateral septum and that the VIP-like neuropeptide serves as a neuromodulator (-transmitter) in this area.     

The Slp4-a linker domain controls exocytosis through interaction with Munc18-1.syntaxin-1a complex

Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a is specifically localized on dense-core vesicles in certain neuroendocrine cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A. However, the precise molecular mechanism of its inhibitory effect on exocytosis has never been elucidated and is still a matter of controversy. Here we show by deletion and chimeric analyses that the linker domain of Slp4-a interacts with the Munc18-1.syntaxin-1a complex by directly binding to Munc18-1 and that this interaction promotes docking of dense-core vesicles to the plasma membrane in PC12 cells. Despite increasing the number of plasma membrane docked vesicles, expression of Slp4-a strongly inhibited high-KCl-induced dense-core vesicle exocytosis. The inhibitory effect by Slp4-a is absolutely dependent on the linker domain of Slp4-a, because substitution of the linker domain of Slp4-a by that of Slp5 (the closest isoform of Slp4-a that cannot bind the Munc18-1.syntaxin-1a complex) completely abrogated the inhibitory effect. Our findings reveal a novel docking machinery for dense-core vesicle exocytosis: Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with syntaxin-1a in the plasma membrane.     

Morphology and location of dense-core vesicles in the stomatogastric ganglion of the lobster,Panulirus interruptus

The appearance and distribution of dense-core vesicles in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus, were examined using transmission electron microscopy. Following five fixation techniques, three types of dense-core vesicles were identified on the basis of size and morphology. Type-I vesicles are found in a distinct neuronal fiber system that appears to be involved in chemical transmission within the ganglion. Type-II vesicles occur in nerve processes in the ganglion, in major nerve trunks and in the perineural sheath of the nerves and ganglion. Type-III vesicles are present in all neuronal somata of the ganglion. The distinct morphology and location of the three types of vesicles suggest that their functional roles differ. Furthermore, the histochemical, biochemical and physiological data available for the stomatogastric ganglion indicate that Type-I vesicles may store dopamine.     

Atg16L1, an essential factor for canonical autophagy, participates in hormone secretion from PC12 cells independently of autophagic activity

Autophagy is a bulk degradation system in all eukaryotic cells and regulates a variety of biological activities in higher eukaryotes. Recently involvement of autophagy in the regulation of the secretory pathway has also been reported, but the molecular mechanism linking autophagy with the secretory pathway remains largely unknown. Here we show that Atg16L1, an essential protein for canonical autophagy, is localized on hormone-containing dense-core vesicles in neuroendocrine PC12 cells and that knockdown of Atg16L1 causes a dramatic reduction in the level of hormone secretion independently of autophagic activity. We also find that Atg16L1 interacts with the small GTPase Rab33A and that this interaction is required for the dense-core vesicle localization of Atg16L1 in PC12 cells. Our findings indicate that Atg16L1 regulates not only autophagy in all cell types, but also secretion from dense-core vesicles, presumably by acting as a Rab33A effector, in particular cell types.