Difference in distribution of membrane proteins between low- and high-density secretory granules in parotid acinar cells

Secretory granules (SGs) are considered to be generated as immature granules and to mature by condensation of their contents. In this study, SGs of parotid gland were separated into low-, medium-, and high-density granule fractions by Percoll-density gradient centrifugation, since it was proposed that the density corresponds to the degree of maturation. The observation with electron microscopy showed that granules in the three fractions were very similar. The average diameter of high-density granules was a little but significantly larger than that of low-density granules. Although the three fractions contained amylase, suggesting that they are all SGs, distribution of membrane proteins was markedly different. Syntaxin6 and VAMP4 were localized in the low-density granule fraction, while VAMP2 was concentrated in the high-density granule fraction. Immunoprecipitation with anti-syntaxin6 antibody caused coprecipitation of VAMP2 from the medium-density granule fraction without solubilization, but not from Triton X-100-solubilized fraction, while VAMP4 was coprecipitated from both fractions. Therefore, VAMP2 is present on the same granules, but is separated from syntaxin6 and VAMP4, which are expected to be removed from immature granules. These results suggest that the medium-density granules are intermediates from low- to high-density granules, and that the membrane components of SGs dynamically change by budding and fusion during maturation.     

Molecular crowding as a mechanism for tick secretory granule biogenesis

During feeding ticks secrete bioactive components into the host to counter-act its immune and hemostatic defense systems. These bioactive components are stored in secretory granules that are secreted during feeding in an exocrine stimulus-response type of mechanism. All proteins destined for secretion are packaged into these granules during granule biogenesis. Up to date no mechanism for granule biogenesis has been proposed, except to note that biogenesis occurs under conditions of high protein and calcium concentrations in an acidic environment. Previously, the most abundant proteins (TSGPs) found in the salivary glands of the soft tick, Ornithodoros savignyi, were suggested to play a part in granule biogenesis, based on their high abundance. The TSGPs are part of the lipocalin family, of which numerous members have been identified in ticks. We consider here the high concentrations of the TSGPs in salivary glands and what effect this will have on the crowded environment inside the secretory granules. It is shown that the TSGPs occur at concentrations that will lead to molecular crowding of which one result is the non-specific aggregation of components to reduce crowding effects. Aggregation of proteins as a mechanism of granule biogenesis has been proposed before, but not in terms of molecular crowding. We thus propose molecular crowding as the general mechanism of granule biogenesis, in tick secretory granules, but can also be extended to other forms of secretory granules in general.     

PGL proteins self associate and bind RNPs to mediate germ granule assembly in C. elegans

Germ granules are germ lineage-specific ribonucleoprotein (RNP) complexes, but how they are assembled and specifically segregated to germ lineage cells remains unclear. Here, we show that the PGL proteins PGL-1 and PGL-3 serve as the scaffold for germ granule formation in Caenorhabditis elegans. Using cultured mammalian cells, we found that PGL proteins have the ability to self-associate and recruit RNPs. Depletion of PGL proteins from early C. elegans embryos caused dispersal of other germ granule components in the cytoplasm, suggesting that PGL proteins are essential for the architecture of germ granules. Using a structure-function analysis in vivo, we found that two functional domains of PGL proteins contribute to germ granule assembly: an RGG box for recruiting RNA and RNA-binding proteins and a self-association domain for formation of globular granules. We propose that self-association of scaffold proteins that can bind to RNPs is a general mechanism by which large RNP granules are formed.     

SCD6 induces ribonucleoprotein granule formation in trypanosomes in a translation-independent manner,regulated by its Lsm and RGG domains

Ribonucleoprotein (RNP) granules are cytoplasmic, microscopically visible structures composed of RNA and protein with proposed functions in mRNA decay and storage. Trypanosomes have several types of RNP granules, but lack most of the granule core components identified in yeast and humans. The exception is SCD6/Rap55, which is essential for processing body (P-body) formation. In this study, we analyzed the role of trypanosome SCD6 in RNP granule formation. Upon overexpression, the majority of SCD6 aggregates to multiple granules enriched at the nuclear periphery that recruit both P-body and stress granule proteins, as well as mRNAs. Granule protein composition depends on granule distance to the nucleus. In contrast to findings in yeast and humans, granule formation does not correlate with translational repression and can also take place in the nucleus after nuclear targeting of SCD6. While the SCD6 Lsm domain alone is both necessary and sufficient for granule induction, the RGG motif determines granule type and number: the absence of an intact RGG motif results in the formation of fewer granules that resemble P-bodies. The differences in granule number remain after nuclear targeting, indicating translation-independent functions of the RGG domain. We propose that, in trypanosomes, a local increase in SCD6 concentration may be sufficient to induce granules by recruiting mRNA. Proteins that bind selectively to the RGG and/or Lsm domain of SCD6 could be responsible for regulating granule type and number.     

The ultrastructure of guinea pig heterophil granulocytes and the heterogeneity of the granules

Summary The development of the heterophil granulocytes in the bone marrow of the guinea pig is described. During the maturation of these cells, three types of granule are formed, not only the azurophil and specific granules already described in other mammals but also a third type of granule referred to here as the nucleated granule. During the process of maturation of the cells, these three types of granule are formed successively. On this basis, two steps can be distinguished in the promyelocyte phase in which primary (nucleated and azurophil) granules are formed, i.e. an early and a late stage, nucleated granules being formed in early and azurophil granules in late promyelocytes. Secondary (specific) granules occur first in myelocytes. In mature heterophils of the guinea pig the granule population is composed of about 85% secondary granules, about 10% azurophil granules, and about 5% nucleated granules. The changes in the granule population during the maturation process were quantified. The observations and calculations point to the occurrence of three mitoses: one in the early and one in the late promyelocyte and the third in the myelocyte.     

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.     

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.     

Regulated proteolysis by cortical granule serine protease 1 at fertilization

Cortical granules are specialized organelles whose contents interact with the extracellular matrix of the fertilized egg to form the block to polyspermy. In sea urchins, the granule contents form a fertilization envelope (FE), and this construction is critically dependent upon protease activity. An autocatalytic serine protease, cortical granule serine protease 1 (CGSP1), has been identified in the cortical granules of Strongylocentrotus purpuratus eggs, and here we examined the regulation of the protease activity and tested potential target substrates of CGSP1. We found that CGSP1 is stored in its full-length, enzymatically quiescent form in the granule, and is inactive at pH 6.5 or below. We determined the pH of the cortical granule by fluorescent indicators and micro-pH probe measurements and found the granules to be pH 5.5, a condition inhibitory to CGSP1 activity. Exposure of the protease to the pH of seawater (pH 8.0) at exocytosis immediately activates the protease. Activation of eggs at pH 6.5 or lower blocks activation of the protease and the resultant FE phenotypes are indistinguishable from a protease-null phenotype. We find that native cortical granule targets of the protease are beta-1,3 glucanase, ovoperoxidase, and the protease itself, but the structural proteins of the granule are not proteolyzed by CGSP1. Whole mount immunolocalization experiments demonstrate that inhibition of CGSP1 activity affects the localization of ovoperoxidase but does not alter targeting of structural proteins to the FE. The mistargeting of ovoperoxidase may lead to spurious peroxidative cross-linking activity and contribute to the lethality observed in protease-null cells. Thus, CGSP1 is proteolytically active only when secreted, due to the low pH of the cortical granules, and it has a small population of targets for cleavage within the cortical granules.     

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.     

Not all secretory granules are created equal: Partitioning of soluble content proteins

Secretory granules carrying fluorescent cargo proteins are widely used to study granule biogenesis, maturation, and regulated exocytosis. We fused the soluble secretory protein peptidylglycine alpha-hydroxylating monooxygenase (PHM) to green fluorescent protein (GFP) to study granule formation. When expressed in AtT-20 or GH3 cells, the PHM-GFP fusion protein partitioned from endogenous hormone (adrenocorticotropic hormone, growth hormone) into separate secretory granule pools. Both exogenous and endogenous granule proteins were stored and released in response to secretagogue. Importantly, we found that segregation of content proteins is not an artifact of overexpression nor peculiar to GFP-tagged proteins. Neither luminal acidification nor cholesterol-rich membrane microdomains play essential roles in soluble content protein segregation. Our data suggest that intrinsic biophysical properties of cargo proteins govern their differential sorting, with segregation occurring during the process of granule maturation. Proteins that can self-aggregate are likely to partition into separate granules, which can accommodate only a few thousand copies of any content protein; proteins that lack tertiary structure are more likely to distribute homogeneously into secretory granules. Therefore, a simple "self-aggregation default" theory may explain the little acknowledged, but commonly observed, tendency for both naturally occurring and exogenous content proteins to segregate from each other into distinct secretory granules.     

3-Methylcholanthrene elicits DNA adduct formation in the CYP1A1 promoter region and attenuates reporter gene expression in rat H4IIE cells

Since it was reported that components of immature secretory granules (ISGs) are different from those of mature secretory granules (MSGs) in rat parotid acinar cells, we have been considering that components of secretory granules (SGs) change dynamically during granule maturation. As the first step to understand the mechanism of granule maturation, we separated low-density detergent-resistant membrane fractions (DRMs) from purified SGs of rat parotid gland. When SGs were lysed by the detergent Brij-58, syntaxin6 and VAMP4 were found in DRMs that were different from the GM1a-rich DRMs containing VAMP2. Because syntaxin6 and VAMP4 are known to be related to granule formation, we attempted to separate DRMs from ISGs. To enrich for ISGs, glands were removed from rats 5h after intraperitoneal injection of isoproterenol and used to purify the newly synthesized granules. Compared to mature granules prepared without injection, these newly formed granules were lower in density and contained higher concentrations of syntaxin6, VAMP4, and gamma-adaptin. This composition is consistent with the characterizations of ISGs. DRMs isolated from the newly formed granules were GM1a-rich and contained syntaxin6, VAMP4, and VAMP2 together. Thus, our findings suggest that syntaxin6 and VAMP4 associate with a GM1a-rich membrane microdomain during granule formation but enter a separate membrane microdomain before transport from granules during maturation.     

Darinaparsin is a multivalent chemotherapeutic which induces incomplete stress response with disruption of microtubules and Shh signaling

Chemotherapeutics and other pharmaceuticals are common sources of cellular stress. Darinaparsin (ZIO-101) is a novel organic arsenical under evaluation as a cancer chemotherapeutic, but the drug's precise mechanism of action is unclear. Stress granule formation is an important cellular stress response, but the mechanisms of formation, maintenance, and dispersal of RNA-containing granules are not fully understood. During stress, small, diffuse granules initially form throughout the cytoplasm. These granules then coalesce near the nucleus into larger granules that disperse once the cellular stress is removed. Complete stress granule formation is dependent upon microtubules. Human cervical cancer (HeLa) cells, pre-treated with nocodazole for microtubule depolymerization, formed only small, diffuse stress granules upon sodium arsenite treatment. Darinaparsin, as a single agent, also induced the formation of small, diffuse stress granules, an effect similar to that of the combination of nocodazole with sodium arsenite. Darinaparsin inhibited the polymerization of microtubules both in vivo and in vitro. Interestingly, upon removal of darinaparsin, the small, diffuse stress granules completed formation with coalescence in the perinuclear region prior to disassembly. These results indicate that RNA stress granules must complete formation prior to disassembly, and completion of stress granule formation is dependent upon microtubules. Finally, treatment of cells with darinaparsin led to a reduction in Sonic hedgehog (Shh) stimulated activation of Gli1 and a loss of primary cilia. Therefore, darinaparsin represents a unique multivalent chemotherapeutic acting on stress induction, microtubule polymerization, and Shh signaling.     

The adrenal paraneurone: tubulin organization

When chromaffin cells from the bovine adrenal medulla are maintained in culture, they develop neuritelike processes which end with growth-cone-like structures. Chromaffin granules were found to migrate from the cell body to the neurite endings. Thus, the intracellular transport of secretory granules, existing in vivo, seems to occur in an exaggerated way in the cultured cells. These cells offer an excellent model for studying the mechanism of transport, particularly the role of microtubules. By immunofluorescent staining, we observed that tubulin antibodies decorate a complex network visible along the neurites. Colchicine treatment induced the disappearance of this network followed by a return of granules in the cell body and a retraction of neurites. To test the presence of tubulin in the chromaffin granule membrane, we used two-dimensional gel electrophoresis and a radioimmunoassay. Our results indicate that tubulin is not a significant component of chromaffin granules. However, binding experiments show that granule membranes are able to bind tubulin through high affinity binding sites. These results show that microtubules appear involved in neurite formation and probably in granule transport. Tubulin is not an integral constituent of the granule membrane, but is present as a result of a reversible specific binding. This insertion of tubulin into the membrane might represent a step in the association between microtubules and secretory granules.     

Large-scale purification of calf pancreatic zymogen granule membranes.

A protocol for isolating milligram quantities of highly purified zymogen granule membranes from calf pancreas was developed. The method provides a fivefold enriched zymogen granule fraction that is virtually free from major isodense contaminants, such as mitochondria and erythrocytes. Isolated granules are osmotically stable in isosmotic KCl buffers with half-lives between 90 and 120 min. They display specific ion permeabilities that can be demonstrated using ionophore probes to override intrinsic control mechanisms. A Cl- conductance, a Cl-/anion exchanger, and a K+ conductance are found in the zymogen granule membrane, as previously reported for rat pancreatic, rat parotid zymogen granules, and rabbit pepsinogen granules. Lysis of calf pancreatic secretory granules in hypotonic buffers and subsequent isolation of pure zymogen granule membranes yield about 5-10 mg membrane protein from approximately 1000 ml pancreas homogenate. The purified zymogen granule membranes are a putative candidate for the rapid identification and purification of epithelial Cl- channels and regulatory proteins, since they contain fewer proteins than plasma membranes.     

PHB颗粒在红豆草根瘤细菌发育中的动态变化

红豆草根瘤胞间隙和侵入线中另有个别细菌含有PHB颗粒,而且数量很少,一个细菌通常仅有一个。随着细菌被从侵入线中释放到寄主细胞中,这些PHB颗粒立即消失。幼龄细菌不含PHB颗粒,成熟细菌一般也不含这种内含物。当细菌衰老时,它们又再度出现,并大量增加,而后很快减少,直至完全消失。从未发现这种颗粒存在于解体细菌中,尽管它们处于各种不同的解体状态。PHB颗粒在细菌发育中的变化表明,它的多少不仅与根瘤细菌发育密切有关,而且也受制于根瘤品种。     

Protein sorting among two distinct export pathways occurs from the content of maturing exocrine storage granules

We have developed a method for separating purified parotid secretory granules according to their degree of maturation, and we have used this method to examine the relationship between granule formation and stimulus-independent (constitutive) protein secretion. Constitutive export of pulse-labeled secretory proteins occurs almost entirely after their appearance in newly formed granules, and this secretion can be resolved kinetically into two distinct components. Later-phase secretion is the more prominent component and, according to kinetic and compositional criteria, appears to result from basal exocytosis of mature granules. In contrast, early-phase secretion (1.5-15% of constitutive protein output) appears to originate from maturing granules but differs significantly from granule content in composition; that is, the early component exports individual protein species in different relative amounts. Maturing granules, which are labeled most highly before and during the appearance of early-phase secretion, possess numerous coated membrane evaginations suggestive of vesicular traffic. We propose that, in addition to basal exocytosis of relatively mature granules, constitutive exocrine secretion results from limited, selective removal of content proteins from forming and maturing granules. Thus protein sorting and packaging occur together in granule compartments. Exocrine secretory granules constitute an extension of the post-Golgi sorting system and are not merely terminal depots for proximally targeted polypeptides.     

The distribution of Concanavalin A receptor sites on the membrane of chromaffin granules.

The distribution of concanavalin A (con A) receptor sites on the membranes of chromaffin granules has been investigated by binding studies using 125I-labelled con A and by electron-microscope studies using ferritin-labelled con A. In both experiments con A was observed to bind to chromaffin granule membranes but not to intact granules. The ferritin-con A particles bind to only one of the two possible surfaces of the chromaffin granule membranes. These results are in agreement with previous observations concerning the asymmetric distribution of saccharide residues on the surfaces of a number of different plasma membranes. They suggest that for the intracellular membrane of the chromaffin granule the saccharide sites, like those in plasma membranes, are not exposed to the cell cytoplasm. Further work is necessary to establish whether these sites are on the inner surface of the membrane or whether they are unmasked during the conversion of granules to membrane ghosts.     

Cellular stress induces cytoplasmic RNA granules in fission yeast

Severe stress causes plant and animal cells to form large cytoplasmic granules containing RNA and proteins. Here, we demonstrate the existence of stress-induced cytoplasmic RNA granules in Schizosaccharomyces pombe. Homologs to several known protein components of mammalian processing bodies and stress granules are found in fission yeast RNA granules. In contrast to mammalian cells, poly(A)-binding protein (Pabp) colocalizes in stress-induced granules with decapping protein. After glucose deprivation, protein kinase A (PKA) is required for accumulation of Pabp-positive granules and translational down-regulation. This is the first demonstration of a role for PKA in RNA granule formation. In mammals, the translation initiation protein eIF2α is a key regulator of formation of granules containing poly(A)-binding protein. In S. pombe, nonphosphorylatable eIF2α does not block but delays granule formation and subsequent clearance after exposure to hyperosmosis. At least two separate pathways in S. pombe appear to regulate stress-induced granules: pka1 mutants are fully proficient to form granules after hyperosmotic shock; conversely, eIF2α does not affect granule formation in glucose starvation. Further, we demonstrate a Pka1-dependent link between calcium perturbation and RNA granules, which has not been described earlier in any organism.     

VAMP8/Endobrevin is a critical factor for the homotypic granule growth in pancreatic acinar cells

The delivery of newly-formed secretory content to the granule inventory occurs through direct fusion of recently formed granules and mature granules. The introduction of knockout mice allowed us to investigate the characteristics of the delivery process and to determine the core protein machinery required for granule growth. The SNARE machinery mediates membrane fusion and is essential for the granule lifecycle. In the current work, we use VAMP8 knockout mice to show that the SNARE machinery plays a critical role in the process of granule homotypic fusion. Consistent with this, the mutated mouse pancreatic acinar secretory granules are significantly smaller compared to the control group, demonstrating few granule profiles that might be the result of homotypic fusion.