Core formation and the acquisition of fusion competence are linked during secretory granule maturation in Tetrahymena

The formation of dense core secretory granules is a multistage process beginning in the trans Golgi network and continuing during a period of granule maturation. Direct interactions between proteins in the membrane and those in the forming dense core may be important for sorting during this process, as well as for organizing membrane proteins in mature granules. We have isolated two mutants in dense core granule formation in the ciliate Tetrahymena thermophila, an organism in which this pathway is genetically accessible. The mutants lie in two distinct genes but have similar phenotypes, marked by accumulation of a set of granule cargo markers in intracellular vesicles resembling immature secretory granules. Sorting to these vesicles appears specific, since they do not contain detectable levels of an extraneous secretory marker. The mutants were initially identified on the basis of aberrant proprotein processing, but also showed defects in the docking of the immature granules. These defects, in core assembly and docking, were similarly conditional with respect to growth conditions, and therefore are likely to be tightly linked. In starved cells, the processing defect was less severe, and the immature granules could dock but still did not undergo stimulated exocytosis. We identified a lumenal protein that localizes to the docking-competent end of wildtype granules, but which is delocalized in the mutants. Our results suggest that dense cores have functionally distinct domains that may be important for organizing membrane proteins involved in docking and fusion.     

TgGRA23, a novel Toxoplasma gondii dense granule protein associated with the parasitophorous vacuole membrane and intravacuolar network

Toxoplasma gondii is an intracellular protozoan parasite, which relies on a specialized compartment, the parasitophorous vacuole (PV), to survive within host cells. Dense granules within the parasite release a large variety of proteins to maintain the integrity of the vacuole structure. Here, we identified a novel dense granule protein in T. gondii, TgGRA23, which is a homolog of the Sarcocystis muris dense granule protein, SmDG32. Recombinant TgGRA23 (rTgGRA23) expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein was used to raise antisera in mice and rabbits. Immunoblotting showed that antisera from the immunized mice and rabbits reacted with parasite lysates to yield a 21-kDa native protein. In addition, immuno-electron microscopic examination showed that TgGRA23 resides in the dense granules, PV membrane and intravacuolar network of the parasite. To confirm the precise subcellular localization of TgGRA23 in T. gondii, an immunofluorescent antibody test was performed using dense granule markers. Notably, TgGRA23 co-localized with other dense granule proteins including TgGRA4 and TgGRA7, in the extracellular-stage parasites. Biochemical experiments indicated that TgGRA23 is insoluble and may form an electrostatic complex that is resistant to non-ionic detergents. Furthermore, specific antibodies to TgGRA23 were detected during the chronic stage of Toxoplasma infection in mice. Our results suggest that TgGRA23 is an as yet unknown member of the T. gondii dense granule proteins, and that it may be involved in remodeling or maintenance of the PV.     

Granule lattice protein 1 (Grl1p), an acidic, calcium-binding protein in Tetrahymena thermophila dense-core secretory granules, influences granule size, shape, content organization, and release but not protein sorting or condensation

The electron-dense cores of regulated secretory granules in the ciliate Tetrahymena thermophila are crystal lattices composed of multiple proteins. Granule synthesis involves a series of steps beginning with protein sorting, followed by the condensation and precise geometric assembly of the granule cargo. These steps may to various degrees be determined by the cargo proteins themselves. A prominent group of granule proteins, in ciliates as well as in vertebrate neuronal and endocrine cells, are acidic, heat-stable, and bind calcium. We focused on a protein with these characteristics named granule lattice protein 1 (Grl1p), which represents 16% of total granule contents, and we have now cloned the corresponding gene. Mutants in which the macronuclear copies of GRL1 have been disrupted continue to synthesize dense-core granules but are nonetheless defective in regulated protein secretion. To understand the nature of this defect, we characterized mutant and wild-type granules. In the absence of Grl1p, the sorting of the remaining granule proteins appears normal, and they condense to form a well-defined core. However, the condensed cores do not demonstrate a visible crystalline lattice, and are notably different from wild type in size and shape. The cellular secretion defect arises from failure of the aberrant granule cores to undergo rapid expansion and extrusion after exocytic fusion of the granule and plasma membranes. The results suggest that sorting, condensation, and precise granule assembly are distinct in their requirements for Grl1p.     

Secretion of Polypeptide Crystals from Tetrahymena thermophila Secretory Organelles (Mucocysts) Depends on Processing by a Cysteine Cathepsin,Cth4p

In many organisms, sophisticated mechanisms facilitate release of peptides in response to extracellular stimuli. In the ciliate Tetrahymena thermophila, efficient peptide secretion depends on specialized vesicles called mucocysts that contain dense crystalline cores that expand rapidly during exocytosis. Core assembly depends of endoproteolytic cleavage of mucocyst proproteins by an aspartyl protease, cathepsin 3 (CTH3). Here, we show that a second enzyme identified by expression profiling, Cth4p, is also required for processing of proGrl proteins and for assembly of functional mucocysts. Cth4p is a cysteine cathepsin that localizes partially to endolysosomal structures and appears to act downstream of, and may be activated by, Cth3p. Disruption of CTH4 results in cells (Δcth4) that show aberrant trimming of Grl proproteins, as well as grossly aberrant mucocyst exocytosis. Surprisingly, Δcth4 cells succeed in assembling crystalline mucocyst cores. However, those cores do not undergo normal directional expansion during exocytosis, and they thus fail to efficiently extrude from the cells. We could phenocopy the Δcth4 defects by mutating conserved catalytic residues, indicating that the in vivo function of Cth4p is enzymatic. Our results indicate that as for canonical proteins packaged in animal secretory granules, the maturation of mucocyst proproteins involves sequential processing steps. The Δcth4 defects uncouple, in an unanticipated way, the assembly of mucocyst cores and their subsequent expansion and thereby reveal a previously unsuspected aspect of polypeptide secretion in ciliates.     

Freeze-fracture study of the rat parathyroid gland under hypo- and hypercalcemic conditions,with special reference to secretory granules

Summary Freeze-fracture images of the parenchymal cells in the parathyroid gland of rats were observed after vitamin D₂ plus calcium chloride-suppression and EGTA-activation of secretion. In cells of the suppressed glands, large bulges protruded from the Golgi cisternae, and large granules with a stalk, which are identified as storage granules, suggest that, during maturation, some storage granules may be connected by long tubules with the Golgi cisternae and supplied with secretory products from the Golgi cisternae via these tubules.In the activated glands, presumptive exocytotic and endocytotic specializations of intramembranous particles of the parenchymal cell plasma membrane were frequently observed. In addition, elevations and complementary shallow depressions of various shape and extent were occasionally encountered in the intercellular space. From their morphological characteristics it was concluded that these originated from secretory granule cores, which are discharged from the parenchymal cells into the intercellular space by exocytosis, and it was suggested that discharged granule cores may retain their spherical shape until they fuse to form a flat conglomerate.     

Maturation of dense core granules in wild type and mutant Tetrahymena thermophila.

Tetrahymena thermophila, a ciliated protozoan, has a well-developed pathway of regulated secretion from dense core granules called mucocysts. Since exocytosis-defective mutants are available, steps in the biogenesis of dense core granules and their fusion with the plasma membrane may be resolved genetically. To describe the steps in biochemical terms, we have generated antisera against mucocyst content proteins. One antiserum is directed against a calcium binding protein, p40, that is released on stimulation of exocytosis. p40 is shown to associate with an insoluble matrix in mature mucocysts. In addition, the antiserum recognizes a larger protein, p60, that is soluble, is not found in mature mucocysts and is not released on stimulation. Pulse-chase experiments support a precursor-product relationship between p60 and p40. Using these proteins as markers, two mutant Tetrahymena strains defective in exocytosis have been shown to accumulate the putative precursor p60 in organelles that can be distinguished from one another and from wild type mucocysts on the basis of density. The kinetics of appearance of insoluble p40 and the mutant phenotypes suggest a model of mucocyst maturation in which sorting precedes matrix condensation.     

Plasmodium falciparum Exported Protein 1 is localized to dense granules in merozoites

Apical organellar proteins in Plasmodium falciparum merozoites play important roles upon invasion. To date, dense granule, the least studied apical organelle, secretes parasite proteins across the parasitophorous vacuole membrane (PVM) to remodel the infected erythrocyte. Although this phenomenon is key to parasite growth and virulence, only five proteins so far have been identified as dense granule proteins. Further elucidation of dense granule molecule(s) is therefore required. P. falciparum Exported Protein (EXP) 1, previously reported as a parasitophorous vacuole membrane (PVM) protein, is considered essential for parasite growth. In this study, we characterized EXP1 using specific anti-EXP1 antibodies generated by immunization of wheat germ cell-free produced recombinant EXP1. Immunofluorescence microscopy (IFA) demonstrated that EXP1 co-localized with RESA, indicating that the protein is initially localized to dense granules in merozoites, followed by translocation to the PVM. The EXP1 localization in dense granule of merozoites and its translocation to the PVM after invasion of erythrocytes were further confirmed by immunoelectron microscopy. Here, we demonstrate that EXP1 is one of the dense granule proteins in merozoites, which is then transported to the PVM after invasion.     

Insight in the exocytotic process in chromaffin cells: regulation by trimeric and monomeric G proteins

Catecholamine secretion from chromaffin cells has been used for a long time as a general model to study exocytosis of large dense core secretory granules. Permeabilization and microinjection techniques have brought the possibility to dissect at the molecular level the multi-protein machinery involved in this complex physiological process. Regulated exocytosis comprises distinct and sequential steps including the priming of secretory granules, the formation of a docking complex between granules and the plasma membrane and the subsequent fusion of the granule with the plasma membrane. Key proteins involved in the exocytotic machinery have been identified. For instance, SNAREs which participate in the docking events in most intracellular transport steps along the secretory pathway, play a role in exocytosis in both neuronal and endocrine cells. However, in contrast to intracellular transport processes for which the highest fusion efficiency is required after correct targeting of the vesicles, the number of exocytotic events in activated secretory cells needs to be tightly controlled. We describe here the multistep control exerted by heterotrimeric and monomeric G proteins on the progression of secretory granules from docking to fusion and the molecular nature of some of their downstream effectors in neuroendocrine chromaffin cells.     

Calcium negatively regulates secretion from dense granules in Toxoplasma gondii

Apicomplexan parasites including Toxoplasma gondii and Plasmodium spp. manufacture a complex arsenal of secreted proteins used to interact with and manipulate their host environment. These proteins are organised into three principle exocytotic compartment types according to their functions: micronemes for extracellular attachment and motility, rhoptries for host cell penetration, and dense granules for subsequent manipulation of the host intracellular environment. The order and timing of these events during the parasite's invasion cycle dictates when exocytosis from each compartment occurs. Tight control of compartment secretion is, therefore, an integral part of apicomplexan biology. Control of microneme exocytosis is best understood, where cytosolic intermediate molecular messengers cGMP and Ca²⁺ act as positive signals. The mechanisms for controlling secretion from rhoptries and dense granules, however, are virtually unknown. Here, we present evidence that dense granule exocytosis is negatively regulated by cytosolic Ca²⁺, and we show that this Ca²⁺‐mediated response is contingent on the function of calcium‐dependent protein kinases TgCDPK1 and TgCDPK3. Reciprocal control of micronemes and dense granules provides an elegant solution to the mutually exclusive functions of these exocytotic compartments in parasite invasion cycles and further demonstrates the central role that Ca²⁺ signalling plays in the invasion biology of apicomplexan parasites.     

The Protozoan Parasite Toxoplasma gondii Targets Proteins to Dense Granules and the Vacuolar Space Using Both Conserved and Unusual Mechanisms

All known proteins that accumulate in the vacuolar space surrounding the obligate intracellular protozoan parasite Toxoplasma gondii are derived from parasite dense granules. To determine if constitutive secretory vesicles could also mediate delivery to the vacuolar space, T. gondii was stably transfected with soluble Escherichia coli alkaline phosphatase and E. coli β-lactamase. Surprisingly, both foreign secretory reporters were delivered quantitatively into parasite dense granules and efficiently secreted into the vacuolar space. Addition of a glycosylphosphatidylinositol membrane anchor rerouted alkaline phosphatase to the parasite surface. Alkaline phosphatase fused to the transmembrane domain and cytoplasmic tail from the endogenous dense granule protein GRA4 localized to dense granules. The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent upon the cytoplasmic tail, but not upon a tyrosine-based motif within the tail. Alkaline phosphatase fused to the vesicular stomatitis virus G protein transmembrane domain and cytoplasmic tail localized primarily to the Golgi, although staining of dense granules and the intravacuolar network was also detected; truncating the cytoplasmic tail decreased Golgi staining and increased delivery to dense granules but blocked delivery to the intravacuolar network. Targeting of secreted proteins to T. gondii dense granules and the plasma membrane uses general mechanisms identified in higher eukaryotic cells but is simplified and exaggerated in scope, while targeting of secreted proteins beyond the boundaries of the parasite involves unusual sorting events.     

EFFECTS OF IONOPHORE A23187 ON OOCYTES OF COMANTHUS JAPONICA (ECHINODERMATA: CRINOIDEA)*

Micromolar amounts of divalent cation ionophore A23187 stimulate full grown (but unfertilizable) oocytes of Comanthus japonica to undergo a cortical reaction that is incomplete: first, cortical granule contents ejected at exocytosis do not coalesce but remain as individual blebs just outside the oocyte; and, second, about a fourth of the cortical granule population does not undergo exo-cytosis and remains in the cortical cytoplasm. Of the cortical granules remaining in the oocyte, some have unreacted contents and others have morphologically modified contents. Fine structures are compared among unreacted cortical granules, internally-reacted cortical granules, extracellular blebs of cortical granule material and normal fertilization membranes. The comparison strongly suggests that the outer dense layer and inner fibrous layer of the normal fertilization membrane are derived, respectively, from the dense patches and from the matrices of the cortical granules.     

Direct membrane retrieval into large vesicles after exocytosis in sea urchin eggs

At fertilization in sea urchin eggs, elevated cytosolic Ca2+ leads to the exocytosis of 15,000-18,000 1.3-microns-diam cortical secretory granules to form the fertilization envelope. Cortical granule exocytosis more than doubles the surface area of the egg. It is thought that much of the added membrane is retrieved by subsequent endocytosis. We have investigated how this is achieved by activating eggs in the presence of aqueous- and lipid-phase fluorescent dyes. We find rapid endocytosis of membrane into 1.5-microns-diam vesicles starting immediately after cortical granule exocytosis and persisting over the following 15 min. The magnitude of this membrane retrieval can compensate for the changes in the plasma membrane of the egg caused by exocytosis. This membrane retrieval is not stimulated by PMA treatment which activates the endocytosis of clathrin-coated vesicles. When eggs are treated with short wave-length ultraviolet light, cortical granule exocytosis still occurs, but granule cores fail to disperse. After egg activation, large vesicles containing semi-intact cortical granule protein cores are observed. These data together with experiments using sequential pulses of fluid-phase markers support the hypothesis that the bulk of membrane retrieval immediately after cortical granule exocytosis is achieved through direct retrieval into large endocytotic structures.     

A role for myosin 1e in cortical granule exocytosis in Xenopus oocytes

Xenopus oocytes undergo dynamic structural changes during maturation and fertilization. Among these, cortical granule exocytosis and compensatory endocytosis provide effective models to study membrane trafficking. This study documents an important role for myosin 1e in cortical granule exocytosis. Myosin 1e is expressed at the earliest stage that cortical granule exocytosis can be detected in oocytes. Prior to exocytosis, myosin 1e relocates to the surface of cortical granules. Overexpression of myosin 1e augments the kinetics of cortical granule exocytosis, whereas tail-derived fragments of myosin 1e inhibit this secretory event (but not constitutive exocytosis). Finally, intracellular injection of myosin 1e antibody inhibits cortical granule exocytosis. Further experiments identified cysteine string proteins as interacting partners for myosin 1e. As constituents of the membrane of cortical granules, cysteine string proteins are also essential for cortical granule exocytosis. Future investigation of the link between myosin 1e and cysteine string proteins should help to clarify basic mechanisms of regulated exocytosis.     

The color of lactotroph secretory granules stained with FM1-43 depends on dye concentration

When pituitary lactotroph granules undergo exocytosis in the presence of FM1-43, their cores absorb dye and fluoresce brightly. We report that different granules fluoresce with different colors, despite being stained with a single fluorescent dye; emission spectra from individual granules show up to a 25 nm difference between the greenest and reddest granules. We found a correlation between granule color and average fluorescence intensity, suggesting that granule color depends upon dye concentration. We confirmed this in two ways: by increasing FM dye concentration in granules, which red shifted granule color, and by partially photobleaching the FM dye in granules, which green shifted granule color. Increasing stimulation intensity (by increasing KCl concentration) increased the proportion of red granules, indicating that granules exocytosing during intense stimulation bound more dye. This, perhaps, reflects differences in granule core maturation and condensation in which mature granules with condensed cores bind more FM dye but require more intense stimulation to be released. Concentration-dependent color shifts of FM dyes may be useful for monitoring aggregation processes occurring on a size scale smaller than the optical limit.     

Are mechanisms of exocytosis neurotransmitter specific?

In their review, Langley and Grant (1997) investigate the question whether mechanisms of exocytosis are neurotransmitter specific. There is now much evidence that the mechanisms governing the exocytosis of the two principal storage organelles—granules (large dense core vesicles) and electron-lucent vesicles—differ. But much less is known concerning potential differences in the release mechanisms of electron-lucent vesicles that store different types of fast neurotransmitters or of granules in different types of neurons. It is an open question whether there is a unifying control mechanism for the exocytosis of, for example, a peptide-containing granule of a glutamatergic neuron, a chromaffin granule, a noradrenergic granule or a granule from a neurosecretory neuron in the pituitary. The small electron-lucent synaptic vesicles of various kind apparently share common molecular components of regulated release. They carry the calcium sensor synaptotagmin, small GTP-binding proteins of the rab3 group or the v-SNARE synaptobrevin. Nevertheless, there may be differences in the regulatory mechanisms. This concerns the type of calcium channel involved or the absence of some of the presynaptic molecules such as rab3a, synapsin I or the t-SNAREs SNAP-25 or syntaxin from distinct types of neurons or sensory cells.     

Lysosomal sorting receptors are essential for secretory granule biogenesis in Tetrahymena

Secretory granules, such as neuronal dense core vesicles, are specialized for storing cargo at high concentration and releasing it via regulated exocytosis in response to extracellular stimuli. Here, we used expression profiling to identify new components of the machinery for sorting proteins into mucocysts, secretory granule-like vesicles in the ciliate Tetrahymena thermophila. We show that assembly of mucocysts depends on proteins classically associated with lysosome biogenesis. In particular, the delivery of nonaggregated, but not aggregated, cargo proteins requires classical receptors of the sortilin/VPS10 family, which indicates that dual mechanisms are involved in sorting to this secretory compartment. In addition, sortilins are required for delivery of a key protease involved in T. thermophila mucocyst maturation. Our results suggest potential similarities in the formation of regulated secretory organelles between even very distantly related eukaryotes.     

Occurrence of xanthommatin containing pigment granules in the epidermal cells of the silkworm,Bombyx mori

A new type of pigment granule was found in the epidermal cells of the quail mutant of the silkworm, Bombyx mori. Electron microscopic observation shows this granule to be dense and distinct from the translucent pteridine granule. After the granules were isolated by sucrose density gradient centrifugation, the pigment was extracted and identified as xanthommatin.Xanthommatin localizes in the pigment granules binding with a protein. By SDS-polyacrylamide gel electrophoresis, the molecular weight of the pigment protein was estimated to be 13 kDa. The pigment granules may have a role in the biosynthesis and accumulation of xanthommatin.     

Neurosecretory granule formation in ligated axons: additional arguments for a local differentiation from a Golgi apparatus extension

The sorting domain for the different types of granules and small synaptic vesicles in neurosecretion is still largely a matter of debate. Some authors state that an exocytotic process has to precede granule formation. In previous studies, we favoured the idea that neurosecretory packages in terminals are assembled from axonal reticulum membranes simply by differentiation at the axon ending, the axonal reticulum being an extension of the Golgi apparatus. By ligating bovine splenic nerve, a de novo differentiation can be induced. After ligation, granules and granulo-tubular complexes appear. They were immunoreactive for SV2, VMAT2 and synaptobrevin II, which are all known to be highly enriched in large dense granules. Previously the granulo-tubular structures have already been recognized as precursor stadia of neurosecretory granules.It is concluded that at a de novo differentiation, a sorting out and aggregation is taking place of molecules typical for large dense granules. The small dense granules and tubules can be considered unripe, precursor forms of the large dense granules. All this occurs in the absence of signs of exocytosis. The present findings corroborate the view that granule formation occurs via local differentiation at an axon ending.     

Palmitoylation-defective asialoglycoprotein receptors are normal in their cellular distribution and ability to bind ligand,but are defective in ligand uptake and degradation

The examination of insulin exocytosis at the single cell level by conventional electrophysiologic and amperometric methods possesses inherent limitations, and may not accurately reflect the morphologic events of exocytosis of the insulin granule. To overcome some of these limitations, we show by epifluorescent microscopy of a fluorescent dye, FM1-43, its incorporation into the plasma membrane and oncoming insulin granules undergoing exocytosis, and their core proteins. Using this method, we tracked exocytosis in real-time in insulinoma INS-1 and single rat islet beta cells in response to KCl and glucose. We observed both single transient and multi-stepwise increases in membrane FM1-43 fluorescence, suggesting single granule exocytosis as well as sequential and compound exocytosis, respectively. Confocal microscopy of nonpermeabilized cells shows that some of the exocytosed insulin granules labeled by the FM1-43 dye could also be labeled with insulin antibodies, suggesting prolonged openings of the fusion pores and slow dissolution of the granule core proteins on the membrane surface.