Showing papers on "Granule (cell biology) published in 2011"

RNA Granules in Germ Cells

Abstract: "Germ granules" are cytoplasmic, nonmembrane-bound organelles unique to germline. Germ granules share components with the P bodies and stress granules of somatic cells, but also contain proteins and RNAs uniquely required for germ cell development. In this review, we focus on recent advances in our understanding of germ granule assembly, dynamics, and function. One hypothesis is that germ granules operate as hubs for the posttranscriptional control of gene expression, a function at the core of the germ cell differentiation program.

Stress-specific composition, assembly and kinetics of stress granules in Saccharomyces cerevisiae

Abstract: Eukaryotic cells respond to cellular stresses by the inhibition of translation and the accumulation of mRNAs in cytoplasmic RNA–protein (ribonucleoprotein) granules termed stress granules and P-bodies. An unresolved issue is how different stresses affect formation of messenger RNP (mRNP) granules. In the present study, we examine how sodium azide (NaN3), which inhibits mitochondrial respiration, affects formation of mRNP granules as compared with glucose deprivation in budding yeast. We observed that NaN3 treatment inhibits translation and triggers formation of P-bodies and stress granules. The composition of stress granules induced by NaN3 differs from that of glucose-deprived cells by containing eukaryotic initiation factor (eIF)3, eIF4A/B, eIF5B and eIF1A proteins, and by lacking the heterogeneous nuclear RNP (hnRNP) protein Hrp1. Moreover, in contrast with glucose-deprived stress granules, NaN3-triggered stress granules show different assembly rules, form faster and independently from P-bodies and dock or merge with P-bodies over time. Strikingly, addition of NaN3 and glucose deprivation in combination, regardless of the order, always results in stress granules of a glucose deprivation nature, suggesting that both granules share an mRNP remodeling pathway. These results indicate that stress granule assembly, kinetics and composition in yeast can vary in a stress-specific manner, which we suggest reflects different rate-limiting steps in a common mRNP remodeling pathway.

P granules extend the nuclear pore complex environment in the C. elegans germ line

Abstract: The immortal and totipotent properties of the germ line depend on determinants within the germ plasm. A common characteristic of germ plasm across phyla is the presence of germ granules, including P granules in Caenorhabditis elegans, which are typically associated with the nuclear periphery. In C. elegans, nuclear pore complex (NPC)-like FG repeat domains are found in the VASA-related P-granule proteins GLH-1, GLH-2, and GLH-4 and other P-granule components. We demonstrate that P granules, like NPCs, are held together by weak hydrophobic interactions and establish a size-exclusion barrier. Our analysis of intestine-expressed proteins revealed that GLH-1 and its FG domain are not sufficient to form granules, but require factors like PGL-1 to nucleate the localized concentration of GLH proteins. GLH-1 is necessary but not sufficient for the perinuclear location of granules in the intestine. Our results suggest that P granules extend the NPC environment in the germ line and provide insights into the roles of the PGL and GLH family proteins.

The biology and dynamics of mammalian cortical granules

Abstract: Cortical granules are membrane bound organelles located in the cortex of unfertilized oocytes. Following fertilization, cortical granules undergo exocytosis to release their contents into the perivitelline space. This secretory process, which is calcium dependent and SNARE protein-mediated pathway, is known as the cortical reaction. After exocytosis, the released cortical granule proteins are responsible for blocking polyspermy by modifying the oocytes' extracellular matrices, such as the zona pellucida in mammals. Mammalian cortical granules range in size from 0.2 um to 0.6 um in diameter and different from most other regulatory secretory organelles in that they are not renewed once released. These granules are only synthesized in female germ cells and transform an egg upon sperm entry; therefore, this unique cellular structure has inherent interest for our understanding of the biology of fertilization. Cortical granules are long thought to be static and awaiting in the cortex of unfertilized oocytes to be stimulated undergoing exocytosis upon gamete fusion. Not till recently, the dynamic nature of cortical granules is appreciated and understood. The latest studies of mammalian cortical granules document that this organelle is not only biochemically heterogeneous, but also displays complex distribution during oocyte development. Interestingly, some cortical granules undergo exocytosis prior to fertilization; and a number of granule components function beyond the time of fertilization in regulating embryonic cleavage and preimplantation development, demonstrating their functional significance in fertilization as well as early embryonic development. The following review will present studies that investigate the biology of cortical granules and will also discuss new findings that uncover the dynamic aspect of this organelle in mammals.

Physicochemical and structural properties of maize and potato starches as a function of granule size.

Abstract: Chemical composition, molecular structure and organization, and thermal and pasting properties of maize and potato starches fractionated on the basis of granule size were investigated to understand heterogeneity within granule populations. For both starches, lipid, protein, and mineral contents decreased and apparent amylose contents increased with granule size. Fully branched (whole) and debranched molecular size distributions in maize starch fractions were invariant with granule size. Higher amylose contents and amylopectin hydrodynamic sizes were found for larger potato starch granules, although debranched molecular size distributions did not vary. Larger granules had higher degrees of crystallinity and greater amounts of double and single helical structures. Systematic differences in pasting and thermal properties were observed with granule size. Results suggest that branch length distributions in both amylose and amylopectin fractions are under tighter biosynthetic control in potato starch than either molecular size or amylose/amylopectin ratio, whereas all three parameters are controlled during the biosynthesis of maize starch.

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

Abstract: 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.

AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila

Abstract: Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing "glue granules" that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1- and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules.

Wolfram Syndrome 1 Gene (WFS1) Product Localizes to Secretory Granules and Determines Granule Acidification in Pancreatic Beta Cells

Abstract: Wolfram syndrome is an autosomal recessive disorder characterized by juvenile-onset insulin-dependent diabetes mellitus and optic atrophy. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER) resident transmembrane protein. The Wfs1-null mouse exhibits progressive insulin deficiency causing diabetes. Previous work suggested that the function of the WFS1 protein is connected to unfolded protein response and to intracellular Ca(2+) homeostasis. However, its precise molecular function in pancreatic β-cells remains elusive. In our present study, immunofluorescent and electron-microscopic analyses revealed that WFS1 localizes not only to ER but also to secretory granules in pancreatic β-cells. Intragranular acidification was assessed by measuring intracellular fluorescence intensity raised by the acidotrophic agent, 3-[2,4-dinitroanilino]-3'-amino-N-methyldipropyramine. Compared with wild-type β-cells, there was a 32% reduction in the intensity in WFS1-deficient β-cells, indicating the impairment of granular acidification. This phenotype may, at least partly, account for the evidence that Wfs1-null islets have impaired proinsulin processing, resulting in an increased circulating proinsulin level. Morphometric analysis using electron microscopy evidenced that the density of secretory granules attached to the plasma membrane was significantly reduced in Wfs1-null β-cells relative to that in wild-type β-cells. This may be relevant to the recent finding that granular acidification is required for the priming of secretory granules preceding exocytosis and may partly explain the fact that glucose-induced insulin secretion is profoundly impaired in young prediabetic Wfs1-null mice. These results thus provide new insights into the molecular mechanisms of β-cell dysfunction in patients with Wolfram syndrome.

Distribution of Granule Channels, Protein, and Phospholipid in Triticale and Corn Starches as Revealed by Confocal Laser Scanning Microscopy

Abstract: The morphology and microstructure of starch granules from two cultivars of triticale and from normal corn were characterized using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Compared to numerous pores distributed randomly on the surfaces of corn starch granules, markedly fewer pores were observed on the surfaces of starch granules isolated from Pronghorn triticale, and even fewer on the surfaces of starch granules isolated from Ultima triticale. CLSM with fluorescence staining revealed that starch-associated protein was predominately distributed on the granule surface and in the internal channels of both triticale and corn starches. However, after triticale starch was treated with SDS or SO2, the radially oriented, protein-filled internal channels of the granules were observed more frequently and extended to the central region of granules. Phospholipid was located mainly on the granule surface but also in channels and throughout granules in triticale starche...

Effect of high‐pressure microfluidization on the structure of cassava starch granule

Abstract: Microfluidization has been applied to modify starch granules. The study was conducted to investigate the effect of microfluidization on the structure and thermal properties of cassava starch-water suspension (20% w/w). The means of optical microscopy, SEM, FTIR spectroscopy, XRD, and DSC were applied to analyze the changes in microstructure, crystallinity, and thermal property. Microscopy observations revealed that native starch granules were oval, round, and truncated in shape. After the microfluidization treatment, a bigger starch granule was partially gelatinized, and a gel-like structure was formed on a granular surface. No significant difference in XRD patterns of the samples were observed and all samples exhibited A-type allomorph. Crystallinity decreased with the pressure. Sample treated at 150 MPa contains 17.1% crystalline glucan polymer, lower than that of native granules which have crystallinity of about 25.8%. A lower crystallinity means poor order of crystalline glucan polymer structure in starch granules. The disruption of crystalline order within the granule was also observed by FTIR measurement. Thermal analysis using DSC indicated that the microfluidization treatment brought about a significant decrease of melting enthalpy. The gelatinization enthalpy was 12.0 and 3.0 J/g for the native sample and samples treated under the 150 MPa, respectively. The results indicate that high-pressure microfluidization process induced the gelatinization of cassava starch, which is evaluated by a percentage of the degree of gelatinization, due to a pronounced decrease with increasing microfluidizing pressure.

Effects of process parameters on granules properties produced in a high shear granulator

Abstract: Results of a study on the influence of process parameters such as impeller speed, granulation time and binder viscosity on granule strength and properties are reported. A high shear granulator (Cyclomix manufactured by Hosokawa Micron B.V., The Netherlands) has been used to produce granules. Calcium carbonate (Durcal) was used as feed powder and aqueous polyethylene glycol (PEG) as the binder. The dried granules have been analysed for their strength, density and size distribution. The results show that increasing the granulation time has a great affect on granules strength, until an optimum time has been reached. The underlying cause is an increase in granule density. Granules are consolidated more at higher impeller speeds. Moreover, the granule size distribution seems not to be affected significantly by an increase in impeller speed. Granules produced with high binder viscosity have a considerably lower strength, wide strength distribution due to poor dispersion of binder on the powder bed. Binder addition methods have showed no considerable effect on granule strength or on granule size distribution.

A role for glutamate transporters in the regulation of insulin secretion.

Abstract: In the brain, glutamate is an extracellular transmitter that mediates cell-to-cell communication Prior to synaptic release it is pumped into vesicles by vesicular glutamate transporters (VGLUTs) To inactivate glutamate receptor responses after release, glutamate is taken up into glial cells or neurons by excitatory amino acid transporters (EAATs) In the pancreatic islets of Langerhans, glutamate is proposed to act as an intracellular messenger, regulating insulin secretion from β-cells, but the mechanisms involved are unknown By immunogold cytochemistry we show that insulin containing secretory granules express VGLUT3 Despite the fact that they have a VGLUT, the levels of glutamate in these granules are low, indicating the presence of a protein that can transport glutamate out of the granules Surprisingly, in β-cells the glutamate transporter EAAT2 is located, not in the plasma membrane as it is in brain cells, but exclusively in insulin-containing secretory granules, together with VGLUT3 In EAAT2 knock out mice, the content of glutamate in secretory granules is higher than in wild type mice These data imply a glutamate cycle in which glutamate is carried into the granules by VGLUT3 and carried out by EAAT2 Perturbing this cycle by knocking down EAAT2 expression with a small interfering RNA, or by over-expressing EAAT2 or a VGLUT in insulin granules, significantly reduced the rate of granule exocytosis Simulations of granule energetics suggest that VGLUT3 and EAAT2 may regulate the pH and membrane potential of the granules and thereby regulate insulin secretion These data suggest that insulin secretion from β-cells is modulated by the flux of glutamate through the secretory granules

Two modes of lytic granule fusion during degranulation by natural killer cells

Abstract: Lytic granules in cytotoxic lymphocytes, which include T cells and natural killer (NK) cells, are secretory lysosomes that release their content upon fusion with the plasma membrane (PM), a process known as degranulation. Although vesicle exocytosis has been extensively studied in endocrine and neuronal cells, much less is known about the fusion of lytic granules in cytotoxic lymphocytes. Here, we used total internal reflection fluorescence microscopy to examine lytic granules labeled with fluorescently tagged Fas ligand (FasL) in the NK cell line NKL stimulated with phorbol ester and ionomycin and in primary NK cells activated by physiological receptor–ligand interactions. Two fusion modes were observed: complete fusion, characterized by loss of granule content and rapid diffusion of FasL at the PM; and incomplete fusion, characterized by transient fusion pore opening and retention of FasL at the fusion site. The pH-sensitive green fluorescence protein (pHluorin) fused to the lumenal domain of FasL was used to visualize fusion pore opening with a time resolution of 30 ms. Upon incomplete fusion, pHluorin emission lasted several seconds in the absence of noticeable diffusion. Thus, we conclude that lytic granules in NK cells undergo both complete and incomplete fusion with the PM, and propose that incomplete fusion may promote efficient recycling of lytic granule membrane after the release of cytotoxic effector molecules.

Cellular stress induces cytoplasmic RNA granules in fission yeast

Abstract: 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 downregulation. This is the first demonstration of a role for PKA in RNA granule formation. In mammals, the translation initiation protein eIF2a is a key regulator of formation of granules containing poly(A)-binding protein. In S. pombe, nonphosphorylatable eIF2a 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, eIF2a 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.

Plantain and Banana Starches: Granule Structural Characteristics Explain the Differences in Their Starch Degradation Patterns

Abstract: Different banana cultivars were used to investigate the influences of starch granule structure and hydrolases on degradation. The highest degrees of starch degradation were observed in dessert bananas during ripening. Scanning electron microscopy images revealed smooth granule surface in the green stage in all cultivars, except for Mysore. The small and round granules were preferentially degraded in all of the cultivars. Terra demonstrated a higher degree of crystallinity and a short amylopectin chain length distribution, resulting in high starch content in the ripe stage. Amylose content and the crystallinity index were more strongly correlated than the distribution of amylopectin branch chain lengths in banana starches. α- and β-amylase activities were found in both forms, soluble in the pulp and associated with the starch granule. Starch-phosphorylase was not found in Mysore. On the basis of the profile of α-amylase in vitro digestion and the structural characteristics, it could be concluded that the starch of plantains has an arrangement of granules more resistant to enzymes than the starch of dessert bananas.

In situ Raman microscopy of starch granule structures in wild type and ae mutant maize kernels

Abstract: A method developed for in situ imaging of starch granule structure in dry seeds has been applied to compare the starch granule structures found in wild type and ae mutant maize kernels. In the isogenic ae mutant the activity of the starch branching enzyme IIb is inhibited, which gives rise to a high amylose starch. The granule structures in the wild type samples have been found to be homogeneous, whereas those in the ae mutant are grossly heterogeneous within individual granules, between granules within individual cells, and between cells across the endosperm. The level of heterogeneity observed in situ appears to be more marked than that previously reported for studies on isolated ae mutant starches. Iodine/potassium iodide staining and polarised light microscopy have been used together with Raman microscopy, which has allowed high-resolution mapping of the composition and physical state of the structures within the granules, to probe the origins of the heterogeneity of the starch structures. Although the mutation inhibits the activity of the branching enzyme within the granules, and both the composition and level of crystallinity within and between granules is variable, the major origin of the heterogeneity of the granule architecture appears to result from significant changes in the assembly and packaging of the crystalline structures within the granule. It is suggested that this arises due to the mutation of the starch branching enzyme introducing defects into the self-assembly of the crystalline structure, resulting in an accumulation of defects and increased randomisation of the granule structure.

Selective Recapture of Secretory Granule Components After Full Collapse Exocytosis in Neuroendocrine Chromaffin Cells

Abstract: In secretory cells, calcium-regulated exocytosis is rapidly followed by compensatory endocytosis. Neuroendocrine cells secrete hormones and neuropeptides through various modes of exo-endocytosis, including kiss-and-run, cavicapture and full-collapse fusion. During kiss-and-run and cavicapture modes, the granule membrane is maintained in an omega shape, whereas it completely merges with the plasma membrane during full-collapse mode. As the composition of the granule membrane is very different from that of the plasma membrane, a precise sorting process of granular proteins must occur. However, the fate of secretory granule membrane after full fusion exocytosis remains uncertain. Here, we investigated the mechanisms governing endocytosis of collapsed granule membranes by following internalization of antibodies labeling the granule membrane protein, dopamine-β-hydroxylase (DBH) in cultured chromaffin cells. Using immunofluorescence and electron microscopy, we observed that after full collapse, DBH remains clustered on the plasma membrane with other specific granule markers and is subsequently internalized through vesicular structures composed mainly of granule components. Moreover, the incorporation of this recaptured granule membrane into an early endosomal compartment is dependent on clathrin and actin. Altogether, these results suggest that after full collapse exocytosis, a selective sorting of granule membrane components is facilitated by the physical preservation of the granule membrane entity on the plasma membrane.