Biogenesis Of Dense Core Vesicles Research Articles

Tomosyn affects dense core vesicle composition but not exocytosis in mammalian neurons.

Tomosyn is a large, non-canonical SNARE protein proposed to act as an inhibitor of SNARE complex formation in the exocytosis of secretory vesicles. In the brain, tomosyn inhibits the fusion of synaptic vesicles (SVs), whereas its role in the fusion of neuropeptide-containing dense core vesicles (DCVs) is unknown. Here, we addressed this question using a new mouse model with a conditional deletion of tomosyn (Stxbp5) and its paralogue tomosyn-2 (Stxbp5l). We monitored DCV exocytosis at single vesicle resolution in tomosyn-deficient primary neurons using a validated pHluorin-based assay. Surprisingly, loss of tomosyns did not affect the number of DCV fusion events but resulted in a strong reduction of intracellular levels of DCV cargos, such as neuropeptide Y (NPY) and brain-derived neurotrophic factor (BDNF). BDNF levels were largely restored by re-expression of tomosyn but not by inhibition of lysosomal proteolysis. Tomosyn's SNARE domain was dispensable for the rescue. The size of the trans-Golgi network and DCVs was decreased, and the speed of DCV cargo flux through Golgi was increased in tomosyn-deficient neurons, suggesting a role for tomosyns in DCV biogenesis. Additionally, tomosyn-deficient neurons showed impaired mRNA expression of some DCV cargos, which was not restored by re-expression of tomosyn and was also observed in Cre-expressing wild-type neurons not carrying loxP sites, suggesting a direct effect of Cre recombinase on neuronal transcription. Taken together, our findings argue against an inhibitory role of tomosyns in neuronal DCV exocytosis and suggests an evolutionary conserved function of tomosyns in the packaging of secretory cargo at the Golgi.

End-binding protein 1 promotes specific motor-cargo association in the cell body prior to axonal delivery of dense core vesicles

Axonal transport is key to neuronal function. Efficient transport requires specific motor-cargo association in the soma, yet the mechanisms regulating this early step remain poorly understood. We found that EBP-1, the C.elegans ortholog of the canonical-microtubule-end-binding protein EB1, promotes the specific association between kinesin-3/KIF1A/UNC-104 and dense core vesicles (DCVs) prior to their axonal delivery. Using single-neuron, invivo labeling of endogenous cargo and EBs, we observed reduced axonal abundance and reduced secretion of DCV cargo, but not other KIF1A/UNC-104 cargoes, in ebp-1 mutants. This reduction could be traced back to fewer exit events from the cell body, where EBP-1 colocalized with the DCV sorting machinery at the trans Golgi, suggesting that this is the site of EBP-1 function. EBP-1 calponin homology (CH) domain was required for directing microtubule growth on the Golgi, and mammalian EB1 interacted with KIF1A in an EBH-domain-dependent manner. Loss- and gain-of-function experiments suggest a model in which both kinesin-3 binding and guidance of microtubule growth at the trans Golgi by EBP-1 promote motor-cargo association at sites of DCV biogenesis. In support of this model, tethering either EBP-1 or a kinesin-3/KIF1A/UNC-104-interacting domain from an unrelated protein to the Golgi restored the axonal abundance of DCV proteins in ebp-1 mutants. These results uncover an unexpected role for a microtubule-associated protein and provide insights into how specific kinesin-3 cargo is delivered to the axon.

An increase in VGF expression through a rapid, transcription-independent, autofeedback mechanism improves cognitive function

The release of neuropeptides from dense core vesicles (DCVs) modulates neuronal activity and plays a critical role in cognitive function and emotion. The granin family is considered a master regulator of DCV biogenesis and the release of DCV cargo molecules. The expression of the VGF protein (nonacronymic), a secreted neuropeptide precursor that also belongs to the extended granin family, has been previously shown to be induced in the brain by hippocampus-dependent learning, and its downregulation is mechanistically linked to neurodegenerative diseases such as Alzheimer’s disease and other mood disorders. Currently, whether changes in translational efficiency of Vgf and other granin mRNAs may be associated and regulated with learning associated neural activity remains largely unknown. Here, we show that either contextual fear memory training or the administration of TLQP-62, a peptide derived from the C-terminal region of the VGF precursor, acutely increases the translation of VGF and other granin proteins, such as CgB and Scg2, via an mTOR-dependent signaling pathway in the absence of measurable increases in mRNA expression. Luciferase-based reporter assays confirmed that the 3′-untranslated region (3′UTR) of the Vgf mRNA represses VGF translation. Consistently, the truncation of the endogenous Vgf mRNA 3′UTR results in substantial increases in VGF protein expression both in cultured primary neurons and in brain tissues from knock in mice expressing a 3′UTR-truncation mutant encoded by the modified Vgf gene. Importantly, Vgf 3′UTR-truncated mice exhibit enhanced memory performance and reduced anxiety- and depression-like behaviors. Our results therefore reveal a rapid, transcription-independent induction of VGF and other granin proteins after learning that are triggered by the VGF-derived peptide TLQP-62. Our findings suggest that the rapid, positive feedforward increase in the synthesis of granin family proteins might be a general mechanism to replenish DCV cargo molecules that have been released in response to neuronal activation and is crucial for memory function and mood stability.

Rab2 drives axonal transport of dense core vesicles and lysosomal organelles.

Fast axonal transport of neuropeptide-containing dense core vesicles (DCVs), endolysosomal organelles, and presynaptic components is critical for maintaining neuronal functionality. How the transport of DCVs is orchestrated remains an important unresolved question. The small GTPase Rab2 mediates DCV biogenesis and endosome-lysosome fusion. Here, we use Drosophila to demonstrate that Rab2 also plays a critical role in bidirectional axonal transport of DCVs, endosomes, and lysosomal organelles, most likely by controlling molecular motors. We further show that the lysosomal motility factor Arl8 is required as well for axonal transport of DCVs, but unlike Rab2, it is also critical for DCV exit from cell bodies into axons. We also provide evidence that the upstream regulators of Rab2 and Arl8, Ema and BORC, activate these GTPases during DCV transport. Our results uncover the mechanisms underlying axonal transport of DCVs and reveal surprising parallels between the regulation of DCV and lysosomal motility.

Rab2 Drives Axonal Transport of Dense Core Vesicles and Lysosomal Organelles

Long range fast axonal transport of neuropeptide-containing dense core vesicles (DCVs), endolysosomal organelles and presynaptic components is critical for maintaining the functionality of neurons. How the transport of DCVs is orchestrated remains an important unresolved question. The small GTPase Rab2 has previously been shown to mediate DCV biogenesis and endosome-lysosome fusion. Here we use the Drosophila model system to demonstrate that Rab2 also plays a critical role in bidirectional axonal transport of DCVs, endosomes and lysosomal organelles, most likely by controlling molecular motors. We further show that the lysosomal motility factor Arl8 is required as well for axonal transport of DCVs, but unlike Rab2 is also critical for DCV exit from cell bodies into axons. Our results uncover the mechanisms responsible for axonal transport of DCVs and reveal surprising parallels between the regulation of DCVs and lysosomal motility.

Dense-core vesicle biogenesis and exocytosis in neurons lacking chromogranins A and B.

Chromogranin A and B (Cgs) are considered to be master regulators of cargo sorting for the regulated secretory pathway (RSP) and dense‐core vesicle (DCV) biogenesis. To test this, we analyzed the release of neuropeptide Y (NPY)‐pHluorin, a live RSP reporter, and the distribution, number, and appearance of DCVs, in mouse hippocampal neurons lacking expression of CHGA and CHGB genes. qRT‐PCR analysis showed that expression of other granin family members was not significantly altered in CgA/B−/− neurons. As synaptic maturation of developing neurons depends on secretion of trophic factors in the RSP, we first analyzed neuronal development in standardized neuronal cultures. Surprisingly, dendritic and axonal length, arborization, synapse density, and synaptic vesicle accumulation in synapses were all normal in CgA/B−/− neurons. Moreover, the number of DCVs outside the soma, stained with endogenous marker Secretogranin II, the number of NPY‐pHluorin puncta, and the total amount of reporter in secretory compartments, as indicated by pH‐sensitive NPY‐pHluorin fluorescence, were all normal in CgA/B−/− neurons. Electron microscopy revealed that synapses contained a normal number of DCVs, with a normal diameter, in CgA/B−/− neurons. In contrast, CgA/B−/− chromaffin cells contained fewer and smaller secretory vesicles with a smaller core size, as previously reported. Finally, live‐cell imaging at single vesicle resolution revealed a normal number of fusion events upon bursts of action potentials in CgA/B−/− neurons. These events had normal kinetics and onset relative to the start of stimulation. Taken together, these data indicate that the two chromogranins are dispensable for cargo sorting in the RSP and DCV biogenesis in mouse hippocampal neurons.

The dense-core vesicle maturation protein CCCP-1 binds RAB-2 and membranes through its C-terminal domain.

Dense-core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled-coil protein CCCP-1 was identified as a component of the DCV biogenesis pathway in the nematode Caenorhabditis elegans. CCCP-1 binds the small GTPase RAB-2 and colocalizes with it at the trans-Golgi. Here, we report a structure-function analysis of CCCP-1 to identify domains of the protein important for its localization, binding to RAB-2, and function in DCV biogenesis. We find that the CCCP-1 C-terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP-1 localization and for binding to RAB-2, and is required for the function of CCCP-1 in DCV biogenesis. In addition, CCCP-1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid-binding motif. We conclude that CCCP-1 is a coiled-coil protein that binds an activated Rab and localizes to the Golgi via its C-terminus, properties similar to members of the golgin family of proteins. CCCP-1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.

CAPS-1 requires its C2, PH, MHD1 and DCV domains for dense core vesicle exocytosis in mammalian CNS neurons

CAPS (calcium-dependent activator protein for secretion) are multi-domain proteins involved in regulated exocytosis of synaptic vesicles (SVs) and dense core vesicles (DCVs). Here, we assessed the contribution of different CAPS-1 domains to its subcellular localization and DCV exocytosis by expressing CAPS-1 mutations in four functional domains in CAPS-1/-2 null mutant (CAPS DKO) mouse hippocampal neurons, which are severely impaired in DCV exocytosis. CAPS DKO neurons showed normal development and no defects in DCV biogenesis and their subcellular distribution. Truncation of the CAPS-1 C-terminus (CAPS Δ654-1355) impaired CAPS-1 synaptic enrichment. Mutations in the C2 (K428E or G476E) or pleckstrin homology (PH; R558D/K560E/K561E) domain did not. However, all mutants rescued DCV exocytosis in CAPS DKO neurons to only 20% of wild type CAPS-1 exocytosis capacity. To assess the relative importance of CAPS for both secretory pathways, we compared effect sizes of CAPS-1/-2 deficiency on SV and DCV exocytosis. Using the same (intense) stimulation, DCV exocytosis was impaired relatively strong (96% inhibition) compared to SV exocytosis (39%). Together, these data show that the CAPS-1 C-terminus regulates synaptic enrichment of CAPS-1. All CAPS-1 functional domains are required, and the C2 and PH domain together are not sufficient, for DCV exocytosis in mammalian CNS neurons.

Role of clathrin in dense core vesicle biogenesis.

The dense core vesicles (DCVs) of neuroendocrine cells are a rich source of bioactive molecules such as peptides, hormones, and neurotransmitters, but relatively little is known about how they are formed. Using fractionation profiling, a method that combines subcellular fractionation with mass spectrometry, we identified ∼1200 proteins in PC12 cell vesicle-enriched fractions, with DCV-associated proteins showing distinct profiles from proteins associated with other types of vesicles. To investigate the role of clathrin in DCV biogenesis, we stably transduced PC12 cells with an inducible short hairpin RNA targeting clathrin heavy chain, resulting in ∼85% protein loss. DCVs could still be observed in the cells by electron microscopy, but mature profiles were approximately fourfold less abundant than in mock-treated cells. By quantitative mass spectrometry, DCV-associated proteins were found to be reduced approximately twofold in clathrin-depleted cells as a whole and approximately fivefold in vesicle-enriched fractions. Our combined data sets enabled us to identify new candidate DCV components. Secretion assays revealed that clathrin depletion causes a near-complete block in secretagogue-induced exocytosis. Taken together, our data indicate that clathrin has a function in DCV biogenesis beyond its established role in removing unwanted proteins from the immature vesicle.

DCVs biogenesis control during secretory activity: transcriptional regulation of FMRF like neuropeptides genes (flps) by neuronal activity in C. elegans

Event Abstract Back to Event DCVs biogenesis control during secretory activity: transcriptional regulation of FMRF like neuropeptides genes (flps) by neuronal activity in C. elegans Juan R. Lorenzo Lopez1, 2*, Aikaterini Stratigi1, 2 and Patrick Laurent1, 2* 1 Université Libre de Bruxelles, Laboratory of Neurophysiology, Faculty of Medicine, Belgium 2 Université libre de Bruxelles, ULB Institute for Neuroscience (UNI), Belgium Neurons and endocrine cells have a regulated secretory pathway in which neuropeptide and peptide hormones are loaded in large dense-core vesicles (DCVs) to be secreted at the cell surface. Because DCVs require de novo biogenesis to replenish the stores and maintain the peptide load, one of the main questions of DCV biology is how the cell controls the expression of proteins needed for DCV production. The C. elegans model has proven to be extremely useful to explore the cell biology of the classical synaptic vesicle offering similar opportunities to investigate the neuropeptide/DCVs biology. The host laboratory recently reported in C. elegans a coupling between neuronal secretory activity and transcriptional activity of several DCVs genes, including flp-5, flp-11, flp-17, flp-21. We tested the activity of flp genes in mutant strains for candidate genes potentially involved in the coupling of cell-activity/exocytosis with neuropeptide transcription. Here, we present our recent findings and discuss a new strategy to test the transcriptional regulation by neuronal activity in C. elegans neurons. Acknowledgements This research is funded by the Wiener-Anspach Foundation (http://fwa.ulb.ac.be). AS is fellow of the Fonds pour la Recherche dans l'Industrie et l'Agriculture (FRIA). PL is Principal Researcher at the FRS-FNRS. References Borgonovo B, Ouwendijk J, Solimena M. Biogenesis of secretory granules. Current opinion in cell biology (2006) 18:365-70. Laurent P, Soltesz Z, Nelson GM, Chen C, Arellano-Carbajal F, Levy E, de Bono M. Decoding a neural circuit controlling global animal state in C. elegans. Elife (2015) 4:e04241. doi: 10.7554/eLife.04241 Uhl GR, Nishimori T. Neuropeptide gene expression and neural activity: assessing a working hypothesis in nucleus caudalis and dorsal horn neurons expressing preproenkephalin and preprodynorphin. T. Cell Mol Neurobiol (1990) 10: 73. doi:10.1007/BF007336 Keywords: dense core vesicle, FMRF like neuropeptides, Transcriptional regulation, Neuronal activity, c elegans Conference: 12th National Congress of the Belgian Society for Neuroscience, Gent, Belgium, 22 May - 22 May, 2017. Presentation Type: Poster Presentation Topic: Neural Excitability, Synapses, and Glia: Cellular Mechanisms Citation: Lorenzo Lopez JR, Stratigi A and Laurent P (2019). DCVs biogenesis control during secretory activity: transcriptional regulation of FMRF like neuropeptides genes (flps) by neuronal activity in C. elegans. Front. Neurosci. Conference Abstract: 12th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2017.94.00025 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 01 May 2017; Published Online: 25 Jan 2019. * Correspondence: PhD. Juan R Lorenzo Lopez, Université Libre de Bruxelles, Laboratory of Neurophysiology, Faculty of Medicine, Brussels, Belgium, lorenzolopezjr@gmail.com PhD. Patrick Laurent, Université Libre de Bruxelles, Laboratory of Neurophysiology, Faculty of Medicine, Brussels, Belgium, patlaure@ulb.ac.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Juan R Lorenzo Lopez Aikaterini Stratigi Patrick Laurent Google Juan R Lorenzo Lopez Aikaterini Stratigi Patrick Laurent Google Scholar Juan R Lorenzo Lopez Aikaterini Stratigi Patrick Laurent PubMed Juan R Lorenzo Lopez Aikaterini Stratigi Patrick Laurent Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles.

The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.

CAPS-1 promotes fusion competence of stationary dense-core vesicles in presynaptic terminals of mammalian neurons.

Neuropeptides released from dense-core vesicles (DCVs) modulate neuronal activity, but the molecules driving DCV secretion in mammalian neurons are largely unknown. We studied the role of calcium-activator protein for secretion (CAPS) proteins in neuronal DCV secretion at single vesicle resolution. Endogenous CAPS-1 co-localized with synaptic markers but was not enriched at every synapse. Deletion of CAPS-1 and CAPS-2 did not affect DCV biogenesis, loading, transport or docking, but DCV secretion was reduced by 70% in CAPS-1/CAPS-2 double null mutant (DKO) neurons and remaining fusion events required prolonged stimulation. CAPS deletion specifically reduced secretion of stationary DCVs. CAPS-1-EYFP expression in DKO neurons restored DCV secretion, but CAPS-1-EYFP and DCVs rarely traveled together. Synaptic localization of CAPS-1-EYFP in DKO neurons was calcium dependent and DCV fusion probability correlated with synaptic CAPS-1-EYFP expression. These data indicate that CAPS-1 promotes fusion competence of immobile (tethered) DCVs in presynaptic terminals and that CAPS-1 localization to DCVs is probably not essential for this role.

A Novel Sorting Prevention Mechanism Mediated by HID-1 during Early Biogenesis of Dense Core Vesicles

Peptide hormones and neuropeptides are packaged and stored in a specialized intracellular organelle called secretory granules (SGs, also known as dene core vesicles, DCV). The molecular mechanisms involved in DCV biogenesis from the trans-Golgi network (TGN) are largely unknown. It is unclear how cargo proteins are correctly sorted or maintained in SGs. In search for mutants with high-temperature-induced dauer formation (Hid) phenotype in C. elegans, a gene designated hid-1 was identified. The hid-1 gene encodes a highly conserved protein (HID-1) with homologues in Drosophila melanogaster, mouse and Homo sapiens. Interestingly, the Hid phenotype of hid-1 mutants is strongly suppressed in C. elegans by mutations in daf-16 gene, which encodes a transcription factor downstream of insulin signalling, suggesting a possible role for HID-1 in the insulin branch of the dauer pathway. Our studies have implicated that HID-1 is involved in the early stages of SG exocytosis. We demonstrated that HID-1 serve as the ‘stop sign’ to prevent excessive mis-sorting of soluble SG cargoes to lysosomes. We provided evidence that this preventive mechanism is essential for the maturation of SGs and peptide hormone signaling.

HID-1, a New Component of the Peptidergic Signaling Pathway

hid-1 was originally identified as a Caenorhabditis elegans gene encoding a novel conserved protein that regulates the decision to enter into the enduring dauer larval stage. We isolated a novel allele of hid-1 in a forward genetic screen for mutants mislocalizing RBF-1 rabphilin, a RAB-27 effector. Here we demonstrate that HID-1 functions in the nervous system to regulate neuromuscular signaling and in the intestine to regulate the defecation motor program. We further show that a conserved N-terminal myristoylated motif of both invertebrate and vertebrate HID-1 is essential for its association with intracellular membranes in nematodes and PC12 cells. C. elegans neuronal HID-1 resides on intracellular membranes in neuronal cell somas; however, the kinesin UNC-104 also transports HID-1 to synaptic regions. HID-1 accumulates in the axons of unc-13 and unc-31 mutants, suggesting it is associated with neurosecretory vesicles. Consistent with this, genetic studies place HID-1 in a peptidergic signaling pathway. Finally, a hid-1 null mutation reduces the levels of endogenous neuropeptides and alters the secretion of fluorescent-tagged cargos derived from neuronal and intestinal dense core vesicles (DCVs). Taken together, our findings indicate that HID-1 is a novel component of a DCV-based neurosecretory pathway and that it regulates one or more aspects of the biogenesis, maturation, or trafficking of DCVs.

Versatile roles for myosin Va in dense core vesicle biogenesis and function

The motor protein myosin Va is involved in multiple successive steps in the development of dense-core vesicles, such as in the membrane remodelling during their maturation, their transport along actin filaments and the regulation of their exocytosis. In the present paper, we summarize the current knowledge on the roles of myosin Va in the different steps of dense-core vesicle biogenesis and exocytosis, and compare findings obtained from different cell types and experimental systems.

Impaired dense core vesicle maturation in Caenorhabditis elegans mutants lacking Rab2

Despite a key role for dense core vesicles (DCVs) in neuronal function, there are major gaps in our understanding of DCV biogenesis. A genetic screen for Caenorhabditis elegans mutants with behavioral defects consistent with impaired DCV function yielded five mutations in UNC-108 (Rab2). A genetic analysis showed that unc-108 mutations impair a DCV function unrelated to neuropeptide release that, together with neuropeptide release, fully accounts for the role of DCVs in locomotion. An electron microscopy analysis of DCVs in unc-108 mutants, coupled with quantitative imaging of DCV cargo proteins, revealed that Rab2 acts in cell somas during DCV maturation to prevent the loss of soluble and membrane cargo. In Rab2 null mutants, two thirds of these cargoes move to early endosomes via a PI(3)P-dependent trafficking pathway, whereas aggregated neuropeptides are unaffected. These results reveal how neurons solve a challenging trafficking problem using the most highly conserved animal Rab.

Mouse Chromaffin Cells Have Two Populations of Dense Core Vesicles

The quantal hypothesis states that neurotransmitter is released in discrete packages, quanta, thought to represent the neurotransmitter content of individual vesicles. If true, then vesicle size should influence quantal size. Although chromaffin cells are generally thought to have a single population of secretory vesicles, our electron microscopy analysis suggested two populations as the size distribution was best described as the sum of two Gaussians. The average volume difference was fivefold. To test whether this difference in volume affected quantal size, neurotransmitter release from permeabilized cells exposed to 100 microM Ca2+ was measured with amperometry. Quantal content was bimodally distributed with both large and small events; the distribution of vesicle sizes predicted by amperometry was extremely similar to those measured with electron microscopy. In addition, each population of events exhibited distinct release kinetics. These results suggest that chromaffin cells have two populations of dense core vesicles (DCV) with unique secretory properties and which may represent two distinct synthetic pathways for DCV biogenesis or alternatively they may represent different stages of biosynthesis.

Tetrahymena: the key to the genetic analysis of the regulated pathway of polypeptide secretion?

The ciliated protozoa Tetrahymena thermophila and T. pyriformis have long been popular among molecular biologists as model organisms in which to elucidate the function of telomeres and have been instrumental in the study of ribosomal catalytic activity and the development of ribozymes (1, 2). In a smaller way they have captured the imagination of cell biologists for the remarkable way in which they embody the attributes of multicellular organisms in a single primitive cell (Fig. 1). They move, they have the equivalent of mouth parts to ingest bacteria that they deliver to digestive vacuoles and excrete the residues at their rear ends. They also have an osmoregulatory mechanism incorporated in a tubular-vesicular system that functions not unlike a kidney to permit them to live in saline or hypoosmotic environments (3). What recently has emerged is that they may provide a very powerful genetic tool for elucidation of the molecular mechanisms involved in the regulated secretion of proteins, polypeptide hormones, and neurotransmitters. Regulated exocytosis in metazoans takes a number of distinct forms exemplified by release of the amine neurotransmitters of synaptic vesicles mediated by changes in membrane potential, the fusion of glucose transporter containing vesicles in muscle and fat in response to insulin stimulation, the release of pancreatic digestive enzymes by gut peptides and neuronal stimulation and insulin secretion in response metabolic signals. The SNARE hypothesis (4) points to a common molecular mechanism of exocytosis; nevertheless, fundamental differences remain in the biogenesis of dense core vesicles (DCV) containing polypeptide hormones or digestive enzymes, which form directly from the trans–Golgi network, and the synaptic vesicles and glucose transporter vesicles, which appear to be derived from an early endosome compartment (5). The DCVs cannot be recycled in the same way as synaptic or transporter vesicles because their cargo is derived from the …