Subset Of Granules Research Articles

Two RNA binding proteins, ADAD2 and RNF17, interact to form a heterogeneous population of novel meiotic germ cell granules with developmentally dependent organelle association.

Mammalian male germ cell differentiation relies on complex RNA biogenesis events, many of which occur in non-membrane bound organelles termed RNA germ cell granules that are rich in RNA binding proteins (RBPs). Though known to be required for male germ cell differentiation, we understand little of the relationships between the numerous granule subtypes. ADAD2, a testis specific RBP, is required for normal male fertility and forms a poorly characterized granule in meiotic germ cells. This work aimed to understand the role of ADAD2 granules in male germ cell differentiation by clearly defining their molecular composition and relationship to other granules. Biochemical analyses identified RNF17, a testis specific RBP that forms meiotic male germ cell granules, as an ADAD2-interacting protein. Phenotypic analysis of Adad2 and Rnf17 mutants identified a rare post-meiotic chromatin defect, suggesting shared biological roles. ADAD2 and RNF17 were found to be dependent on one another for granularization and together form a previously unstudied set of germ cell granules. Based on co-localization studies with well-characterized granule RBPs and organelle-specific markers, a subset of the ADAD2-RNF17 granules are found to be associated with the intermitochondrial cement and piRNA biogenesis. In contrast, a second, morphologically distinct population of ADAD2-RNF17 granules co-localized with the translation regulators NANOS1 and PUM1, along with the molecular chaperone PDI. These large granules form a unique funnel-shaped structure that displays distinct protein subdomains and is tightly associated with the endoplasmic reticulum. Developmental studies suggest the different granule populations represent different phases of a granule maturation process. Lastly, a double Adad2-Rnf17 mutant model suggests the interaction between ADAD2 and RNF17, as opposed to loss of either, is the likely driver of the Adad2 and Rnf17 mutant phenotypes. These findings shed light on the relationship between germ cell granule pools and define new genetic approaches to their study.

Munc13b stimulus-dependently accumulates on granuphilin-mediated, docked granules prior to fusion.

The Rab27 effector granuphilin plays an indispensable role in stable docking of secretory granules to the plasma membrane by interacting with the complex of Munc18-1 and the fusion-incompetent, closed form of syntaxins-1~3. Although this process prevents spontaneous granule exocytosis, those docked granules actively fuse in parallel with other undocked granules after stimulation. Therefore, it is postulated that the closed form of syntaxins must be converted into the fusion-competent open form in a stimulus-dependent manner. Although Munc13 family proteins are generally thought to prime docked vesicles by facilitating conformational change in syntaxins, it is unknown which isoform acts in granuphilin-mediated, docked granule exocytosis. In the present study, we show that, although both Munc13a and Munc13b are expressed in mouse pancreatic islets and their beta-cell line MIN6, the silencing of Munc13b, but not that of Munc13a, severely affects glucose-induced insulin secretion. Furthermore, Munc13b accumulates on a subset of granules beneath the plasma membrane just prior to fusion during stimulation, whereas Munc13a is translocated to the plasma membrane where granules do not exist. When fluorescently labeled granuphilin was introduced to discriminate between molecularly docked granules and other undocked granules in living cells, Munc13b downregulation was observed to preferentially decrease the fusion of granuphilin-positive granules immobilized to the plasma membrane. These findings suggest that Munc13b promotes insulin exocytosis by clustering on molecularly docked granules in a stimulus-dependent manner.Key words: docking, insulin, live cell imaging, priming, TIRF microscopy.

The GNU subunit of PNG kinase, the developmental regulator of mRNA translation, binds BIC-C to localize to RNP granules.

Control of mRNA translation is a key mechanism by which the differentiated oocyte transitions to a totipotent embryo. In Drosophila, the PNG kinase complex regulates maternal mRNA translation at the oocyte-to-embryo transition. We previously showed that the GNU activating subunit is crucial in regulating PNG and timing its activity to the window between egg activation and early embryogenesis (Hara et al., 2017). In this study, we find associations between GNU and proteins of RNP granules and demonstrate that GNU localizes to cytoplasmic RNP granules in the mature oocyte, identifying GNU as a new component of a subset of RNP granules. Furthermore, we define roles for the domains of GNU. Interactions between GNU and the granule component BIC-C reveal potential conserved functions for translational regulation in metazoan development. We propose that by binding to BIC-C, upon egg activation GNU brings PNG to its initial targets, translational repressors in RNP granules.

Tunable activation of therapeutic platelet-rich plasma by pulse electric field: Differential effects on clot formation, growth factor release, and platelet morphology.

BackgroundActivation of platelet-rich plasma (PRP) by pulse electric field (PEF) releases growth factors which promote wound healing (e.g., PDGF, VEGF for granulation, EGF for epithelialization).AimsTo determine after PEF activation of therapeutic PRP: 1) platelet gel strength; 2) profile of released growth factors; 3) alpha- and T-granule release; 4) platelet morphology.MethodsConcentrated normal donor PRP was activated by 5 μsec (long) monopolar pulse, ~4000 V/cm (PEF A) or 150 nsec (short) bipolar pulse, ~3000 V/cm (PEF B) in the presence of 2.5 mM (low) or 20 mM (high) added CaCl2. Clot formation was evaluated by thromboelastography (TEG). Surface exposure of alpha granule (P-selectin) and T-granule (TLR9 and protein disulfide isomerase [PDI]) markers were assessed by flow cytometry. Factors in supernatants of activated PRP were measured by ELISA. Platelet morphology was evaluated by transmission electron microscopy (TEM).ResultsTime to initial clot formation was shorter with thrombin (<1 min) than with PEF A and B (4.4–8.7 min) but clot strength (elastic modulus, derived from TEG maximum amplitude) was greater with PEF B than with either thrombin or PEF A (p<0.05). Supernatants of PRP activated with PEF A had higher EGF levels than supernatants from all other conditions. In contrast, levels of PF4, PDGF, and VEGF in supernatants were not significantly different after PEF A, PEF B, and thrombin activation. T-granule markers (TLR9 and PDI) were higher after thrombin than after PEF A or B with low or high CaCl2. By TEM, platelets in PEF-treated samples retained a subset of granules suggesting regulated granule release.ConclusionPulse length and polarity can be modulated to produce therapeutic platelet gels as strong or stronger than those produced by thrombin, and this is tunable to produce growth factor profiles enhanced in specific factors important for different stages of wound healing.

Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging.

During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing unspliced HIV-1 RNA.

A heterogeneous tRNA granule structure exhibiting rapid, bi-directional neuritic transport

mRNA translation is regulated by diverse mechanisms that converge at the initiation and elongation steps to determine the rate, profile, and localization of proteins synthesized. A consistently relevant feature of these mechanisms is the spatial re-distribution of translation machinery, a process of particular importance in neural cells. This process has, however, been largely overlooked with respect to its potential role in regulating the local concentration of cytoplasmic tRNAs, even as a multitude of data suggest that spatial regulation of the tRNA pool may help explain the remarkably high rates of peptide elongation. Here, we report that Cy3/Cy5-labeled bulk tRNAs transfected into neural cells distribute into granule-like structures – “tRNA granules” – that exhibit dynamic mixing of tRNAs between granules and rapid, bi-directional vectorial movement within neurites. Imaging of endogenous tRNAgly and tRNAlys by fluorescent in situ hybridization revealed a similar granular distribution of tRNAs in somata and neurites; this distribution was highly overlapping with granules imaged by introduction of exogenous Cy5-tRNAthr and Cy3-tRNAval. A subset of tRNA granules located in the cell body, neurite branch points and growth cones displayed fluorescence resonance energy transfer (FRET) between Cy3 and Cy5-labeled tRNAs indicative of translation, and co-localization with elongation machinery. A population of smaller, rapidly trafficked granules in neurites lacked FRET and showed poor colocalization with translation initiation and elongation factors, suggesting that they are a translationally inactive tRNA transport particle. Our data suggest that tRNAs are packaged into granules that are rapidly transported to loci where translation is needed, where they may greatly increase the local concentration of tRNAs in support of efficient elongation. The potential implications of this newly described structure for channeling of elongation, local translation, and diseases associated with altered tRNA levels or function are discussed.

Characterization of HAF-4- and HAF-9-localizing organelles as distinct organelles in Caenorhabditis elegans intestinal cells.

BackgroundThe intestinal cells of Caenorhabditis elegans are filled with heterogeneous granular organelles that are associated with specific organ functions. The best studied of these organelles are lipid droplets and acidified gut granules associated with GLO-1, a homolog of the small GTPase Rab38. In this study, we characterized a subset of the intestinal granules in which HAF-4 and HAF-9 localize on the membrane. HAF-4 and HAF-9 are ATP-binding cassette (ABC) transporter proteins that are homologous to the mammalian lysosomal peptide transporter TAPL (transporter associated with antigen processing-like, ABCB9).ResultsUsing transgenic worms expressing fluorescent protein-tagged marker proteins, we demonstrated that the HAF-4- and HAF-9-localizing organelles are not lipid droplets and do not participate in yolk protein transport. They were also ruled out as GLO-1-positive acidified gut granules. Furthermore, we clarified that the late endosomal protein RAB-7 localizes to the HAF-4- and HAF-9-localizing organelles and is required for their biogenesis.ConclusionsOur results indicate that the HAF-4- and HAF-9-localizing organelles are distinct intestinal organelles associated with the endocytic pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-015-0076-2) contains supplementary material, which is available to authorized users.

2013 scientific sessions Sol Sherry distinguished lecture in thrombosis: polyphosphate: a novel modulator of hemostasis and thrombosis.

Polyphosphate is a highly anionic, linear polymer of inorganic phosphates that is found throughout biology, including in many infectious microorganisms. Recently, polyphosphate was discovered to be stored in a subset of the secretory granules of human platelets and mast cells, and to be secreted on activation of these cells. Work from our laboratory and others has now shown that polyphosphate is a novel, potent modulator of the blood clotting and complement systems that likely plays roles in hemostasis, thrombosis, inflammation, and host responses to pathogens. Therapeutics targeting polyphosphate may have the potential to limit thrombosis with fewer hemorrhagic complications than conventional anticoagulant drugs that target essential proteases of the blood clotting cascade.

Modulation of autoimmune diabetes by innate-like B cell derived antibodies specific for N-acetyl-D-glucosamine. (BA7P.148)

Abstract The immunodominant epitope of Group A Streptococcus (GAS) is the cell wall associated N-acetyl-D-Glucosamine (GlcNAc) of Group A Carbohydrate (GAC). Previous reports in humans and rodent models have suggested a link between GAS exposure and protection from T1D. Through neonatal immunization with GAS and serum-transfer experiments we find that T1D protection elicited by neonatal GAS exposure is conferred by the antibody (Ab) response to GAC, and the differentiation of unique GlcNAc-specific clonotypes into long-lived plasma cells. Glycan microarray analysis of GAC-specific antibodies indicates they recognize aberrantly truncated mammalian glycans. GlcNAc-terminating glycans exist on a subset of insulin-containing granules and are generated as neoepitpoes during beta cell apoptosis. We find GAC-specific Abs directly block recognition of GlcNAc-modified autoantigens by innate lectins, dampen inflammatory complement activation, and reduce the capacity of dendritic cells to present beta cell antigen. Importantly, T1D patients are deficient in GAS-reactive IgM antibodies. These observations suggest that early exogenous antigen exposure in crucial for developing protective GlcNAc-specific natural antibodies and validate a role for the hygiene hypothesis in T1D. Subsets of GlcNAc-specific antibodies may serve as biomarkers for identifying at-risk patients with natural antibody deficits, and boosting these clonotypes may serve to suppress T1D development

Domains involved in TAF15 subcellular localisation: Dependence on cell type and ongoing transcription

TAF15 (TBP associated factor 15) is a member of the highly conserved TET (also known as FET) protein family of RNA binding proteins (RBP), which comprises in addition FUS (fused in sarcoma, also known as TLS, translocated in liposarcoma) and EWS (Ewing sarcoma protein). The TET proteins are implied to play important roles in the onset of specific tumours, certain forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In this study we identified the domains of TAF15 responsible for its subcellular localisation in human (HeLa) cells and experimentally confirmed the presence of a transportin‐dependent nuclear localisation signal (NLS) at its carboxy-terminus. We demonstrated that additional domains of TAF15 contributed, albeit to a less prominent extent, to its subcellular localisation. In the carboxy-terminus we identified an arginine and glycine rich (RGG) domain, capable of being targeted to stress granules.We, moreover, showed that TAF15 cellular localisation depended on ongoing transcription and that independent domains of TAF15 engaged in nucleolar capping upon transcription inhibition. Finally, we demonstrated that TAF15 localisation was differentially regulated in the HeLa and the neuronal HT22 cell lines and that TAF15 co-localised with a minor subset of RNA granules in the cytoplasm of HT22 cells, supporting a model whereupon TAF15 plays a role in RNA transport and/or local RNA translation.

Characterizing physicochemical changes of cookies baked in a commercial oven

The objective of this research was to understand the effects of baking in an industrial tunnel oven on the physicochemical properties of commercially formulated short-dough cookies. Quality parameters of the cookies, including moisture content, dimensions and color were tracked throughout baking. Instrumental evaluations of compression and fracturability, acrylamide concentration and proton mobility were conducted and used to explain the changes that occurred. Peak acrylamide values were observed after L* values dropped, suggesting there may be a point at which color development and low acrylamide values overlap, allowing for optimization of color and lower acrylamide content. Pasting properties of cookies as measured by RVA showed few differences. Baking resulted in a loss of birefringence in a subset of A-type starch granules providing some evidence of starch granule damage. Proton mobility decreased during baking as moisture loss progressed and interactions between starch and water increased. Isolation of starch from the treatments however demonstrated few differences in crystallinity, pasting behavior or gelatinization enthalpy. Characterization of these product parameters and process variables could suggest strategies for altering a commercial process to modulate quality and also develop strategies to reduce energy consumption.

Polarized TIRFM Reveals Changes in Plasma Membrane Topology Before and During Granule Fusion

We have recently developed a combination of polarization and total internal reflection fluorescence microscopy (pTIRFM) to monitor changes in plasma membrane topology occurring after fusion of chromaffin granules. In this report, pTIRFM is further exploited to reveal two major findings in regards to the secretory pathway in bovine chromaffin cells. First, we show that changes in membrane topology are sometimes detected even prior to fusion. This occurs with high probability in a small subset of granules that appear in the evanescent field during the experiment. On these occasions, the plasma membrane invaginates with the movement just preceding the appearance of a granule in the evanescent field. Such events may represent a direct interaction of the granule with the plasma membrane. Second, we show that the topological fate of the post-fusion, granule/plasma membrane intermediate is regulated by divalent cation. When Sr2+ is used instead of Ca2+ to trigger exocytosis, membrane topology in the exocytotic region is stabilized with significant curvature and indentation.

Ficolin-1 is present in a highly mobilizable subset of human neutrophil granules and associates with the cell surface after stimulation with fMLP

Ficolins are soluble molecules that bind carbohydrate present on the surface of microorganisms and function as recognition molecules in the lectin complement pathway. Three ficolins have been identified in humans: ficolin-1, ficolin-2, and ficolin-3. Ficolin-1 is synthesized in monocytes and type II alveolar epithelial cells. Ficolin-1 has been shown to be present in secretory granules of human neutrophils, but it is not known which subset of the neutrophils' secretory granules harbors ficolin-1. To determine the exact subcellular localization of ficolin-1 in neutrophils, recombinant ficolin-1 was expressed in Chinese hamster ovary cells and used for generation of polyclonal antibodies. This allowed detection of ficolin-1 in subcellular fractions of human neutrophils by ELISA, by Western blotting, and by immunohistochemistry. Real-time PCR examination of normal human bone marrow showed FCN1 gene expression largely in myelocytes, metamyelocytes, and band cells with a profile quite similar to that of gelatinase. In accordance with this, biosynthesis studies of neutrophils precursor cells showed that ficolin-1 was primarily synthesized in myelocytes, metamyelocytes, and band cells. Immunohistochemistry and subcellular fractionation demonstrated that ficolin-1 is primarily localized in gelatinase granules but also in highly exocytosable gelatinase-poor granules, not described previously. Ficolin-1 is released from neutrophil granules by stimulation with fMLP or PMA, and the majority becomes associated with the surface membrane of the cells and can be detected by flow cytometry. Our studies show that neutrophils are a major source of ficolin-1, which can be readily exocytosed by stimulation.

Expression of Antizyme Inhibitor 2 in Mast Cells and Role of Polyamines as Selective Regulators of Serotonin Secretion

BackgroundUpon IgE-mediated activation, mast cells (MC) exocytose their cytoplasmic secretory granules and release a variety of bioactive substances that trigger inflammatory responses. Polyamines mediate numerous cellular and physiological functions. We report here that MCs express antizyme inhibitor 2 (AZIN2), an activator of polyamine biosynthesis, previously reported to be exclusively expressed in the brain and testis. We have investigated the intracellular localization of AZIN2 both in resting and activated MCs. In addition, we have examined the functional role of polyamines, downstream effectors of AZIN2, as potential regulators of MC activity.Methodology/Principal FindingsImmunostainings show that AZIN2 is expressed in primary and neoplastic human and rodent MCs. We demonstrate that AZIN2 localizes in the Vamp-8 positive, serotonin-containing subset of MC granules, but not in tryptase-containing granules, as revealed by double immunofluorescence stainings. Furthermore, activation of MCs induces rapid upregulation of AZIN2 expression and its redistribution, suggesting a role for AZIN2 in secretory granule exocytosis. We also demonstrate that release of serotonin from activated MCs is polyamine-dependent whereas release of histamine and β-hexosaminidase is not, indicating a granule subtype-specific function for polyamines.Conclusions/SignificanceThe study reports for the first time the expression of AZIN2 outside the brain and testis, and demonstrates the intracellular localization of endogenous AZIN2 in MCs. The granule subtype-specific expression and its induction after MC activation suggest a role for AZIN2 as a local, in situ regulator of polyamine biosynthesis in association with serotonin-containing granules of MCs. Furthermore, our data indicates a novel function for polyamines as selective regulators of serotonin release from MCs.

Normal formation of a subset of intestinal granules in Caenorhabditis elegans requires ATP-binding cassette transporters HAF-4 and HAF-9, which are highly homologous to human lysosomal peptide transporter TAP-like.

TAP-like (TAPL; ABCB9) is a half-type ATP-binding cassette (ABC) transporter that localizes in lysosome and putatively conveys peptides from cytosol to lysosome. However, the physiological role of this transporter remains to be elucidated. Comparison of genome databases reveals that TAPL is conserved in various species from a simple model organism, Caenorhabditis elegans, to mammals. C. elegans possesses homologous TAPL genes: haf-4 and haf-9. In this study, we examined the tissue-specific expression of these two genes and analyzed the phenotypes of the loss-of-function mutants for haf-4 and haf-9 to elucidate the in vivo function of these genes. Both HAF-4 and HAF-9 tagged with green fluorescent protein (GFP) were mainly localized on the membrane of nonacidic but lysosome-associated membrane protein homologue (LMP-1)-positive intestinal granules from larval to adult stage. The mutants for haf-4 and haf-9 exhibited granular defects in late larval and young adult intestinal cells, associated with decreased brood size, prolonged defecation cycle, and slow growth. The intestinal granular phenotype was rescued by the overexpression of the GFP-tagged wild-type protein, but not by the ATP-unbound form of HAF-4. These results demonstrate that two ABC transporters, HAF-4 and HAF-9, are related to intestinal granular formation and some other physiological aspects.

Ficolin-1 Is Present in a Highly Mobilizable Subset of Human Neutrophil Granules and Associates with the Cell Surface after Stimulation with fMLP.

Ficolins are soluble pattern recognition molecules that bind carbohydrate structures on the surface of microorganisms. Three ficolins have been identified in man: ficolin-1, ficolin-2, and ficolin-3. Ficolin-1 is derived from the FCN1 gene on chromosome 9 and is synthesized in monocytes and type II alveolar epithelial cells. Ficolin-1 has been shown to be present in secretory granules of human neutrophils, but which subset of the neutrophils secretory granules harbors ficolin-1 is not known. In order to determine the exact subcellular localization of ficolin-1 in neutrophils, recombinant ficolin-1 was expressed in Chinese hamster ovary cells and used for generation of polyclonal antibodies. This allowed detection of ficolin-1 in subcellular fractions of human neutrophils by ELISA and western blotting, and by immunohistochemistry. Real time PCR examination of normal human bone marrow showed FCN1 gene expression largely in myelocytes, metamyelocytes, and band cells with a profile quite similar to that of gelatinase. In accordance with this, immunohistochemistry and subcellular fractionation demonstrated that ficolin-1 is primarily localized in gelatinase granules, but also in highly exocytosable gelatinase poor granules, not previously described.Ficolin-1 is released from neutrophil granules by stimulation with fMLP or PMA, and a significant part becomes associated with the surface membrane of the cells and can be detected by flow cytometry. Our studies show that neutrophils are a major source of ficolin-1, which can be readily released to the surroundings by stimulation. [Display omitted]

TFF (Trefoil Factor Family) Peptides and their Potential Roles for Differentiation Processes During Airway Remodeling

Several lines of defense maintain the surface integrity of the delicate airway epithelium which is regularly subjected to severe trauma. These defense mechanisms include protection by the mucus layer, rapid repair by restitution (cell migration) and regeneration via proliferation and differentiation. Luminal surveillance peptides such as epidermal growth factor (EGF) and trefoil factor family (TFF) peptides support synergistically these processes. TFFs are well known particularly for their key role in mucosal restitution and there is an increasing body of evidence that TFFs also support mucosal differentiation processes. Mucus overproduction during inflammatory and obstructive airway diseases is a partial consequence of an increase in the number of goblet cells due to cell division (goblet cell hyperplasia) or differentiation (goblet cell metaplasia). Particularly the latter process reflects the plasticity of the airway epithelium and causes intense airway remodeling. Goblet cells are derived, at least in part, from Clara cells, which trans-differentiate from a serous into a mucous phenotype. This process is critically dependent upon IL-13. In a recent report (Kouznetsova et al. AJRCMB 36:286-297, 2007) using a murine asthma model it was shown that trans-differentiating Clara cells specifically express Tff1 which is stored in a specific subset of secretory granules. This points to a role for Tff1 as an autocrine factor for the trans-differentiation of Clara cells toward goblet cells. Such a role of TFFs for differentiation processes of the airways is supported by another recent study (LeSimple et al. AJRCMB 36:296-303, 2007) where induction of TFF3 synthesis was shown with differentiation in in vivo humanized tracheal xenograft and in vitro air-liquid interface culture models. Furthermore, exogenous TFF3 promoted differentiation of ciliated cells in an EGF receptor-dependent manner. Taken together, both studies imply that TFFs may play key roles for various differentiation processes of the airways and they could be promising novel targets in order to treat severe chronic and acute airway diseases.

Myosin-Va Facilitates the Accumulation of mRNA/Protein Complex in Dendritic Spines

mRNA localization has an essential role in localizing cytoplasmic determinants, controlling the direction of protein secretion, and allowing the local control of protein synthesis in neurons. In neuronal dendrites, the localization and translocation of mRNA is considered as one of the molecular bases of synaptic plasticity. Recent imaging and functional studies revealed that several RNA-binding proteins form a large messenger ribonucleoprotein (mRNP) complex that is involved in transport and translation of mRNA in dendrites. However, the mechanism of mRNA translocation into dendritic spines is unknown. Here, we show that an actin-based motor, myosin-Va, plays a significant role in mRNP transport in neuronal dendrites and spines. Myosin-Va was Ca2+-dependently associated with TLS, an RNA-binding protein, and its target RNA Nd1-L, an actin stabilizer. A dominant-negative mutant or RNAi of myosin-Va in neurons suppressed TLS accumulation in spines and further impaired TLS dynamics upon activation of mGluRs. The TLS translocation into spines was impeded also in neurons prepared from myosin-Va-null dilute-lethal (dl) mice, which exhibit neurological defects. Our results demonstrate that myosin-Va facilitates the transport of TLS-containing mRNP complexes in spines and may function in synaptic plasticity through Ca2+ signaling.

Relation of hippocampal phospho-SAPK/JNK granules in Alzheimer’s disease and tauopathies to granulovacuolar degeneration bodies

Protein misfolding is a distinguishing feature of a number of neurodegenerative diseases. Accumulation of misfolded protein often results in cellular lesions, the location of lesions correlating with the nature of symptoms. Alzheimer's disease (AD), Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD) and Pick's Disease (PiD) all present with pathological lesions containing hyperphosphorylated filamentous tau protein; however, the location and type of lesion varies. In addition, granulovacuolar degeneration (GVD) bodies have been reported within hippocampal pyramidal neurons in AD, PSP, CBD and PiD tissue. GVDs are defined as electron-dense granules within double membrane-bound cytoplasmic vacuoles. We have previously reported that the phosphorylated form of stress-activated protein kinase/c-Jun N-terminal kinase (p-SAPK/JNK) accumulates in granules within hippocampal pyramidal cell bodies in AD tissue at the time that hyperphosphorylated tau begins to aggregate into early-stage NFTs. We now report that p-SAPK/JNK granules are found within the hippocampal CA1 region of PSP, CBD and PiD cases as well and that these granules are likely GVD bodies. Quantitatively, p-SAPK/JNK granules and GVDs are found in comparable numbers of CA1 cells. Within cells, p-SAPK/JNK granules are distributed throughout the cytoplasm in a manner similar to the distribution of GVDs and a subset of granules co-localize with GVD markers. Ultrastructurally, p-SAPK/JNK granules are located in large cytoplasmic vacuoles, thereby fitting the definition of a GVD body. With the implication of granular p-SAPK/JNK as a marker of GVDs, our study strongly suggests that a heterogeneous group of proteins form GVDs. The mechanism of GVD formation is therefore an interesting one, and is likely separate and distinct from the mechanism of tau inclusion formation.

The Class I HLA Repertoire of Pancreatic Islets Comprises the Nonclassical Class Ib Antigen HLA-G

Selective expression of the human class Ib HLA molecule HLA-G in immunologically protected sites and its function in the inhibition of NK and T-cell effector functions support an important role of this molecule in immunoregulation. Here, we demonstrate that HLA-G is constitutively expressed in the endocrine compartment of the human pancreas. Surface expression of this HLA determinant in endocrine cells is regulated in response to growth and inflammatory stimuli. Furthermore, we provide evidence that HLA-G expressed in this tissue may associate with a subset of insulin-containing granules and may be shuttled to the cell surface in response to secretory stimuli. Thus, HLA-G presentation by endocrine cells may be regulated in concert with their secretory activity. These results identify the expression of a major histocompatibility complex locus with putative regulatory functions in human pancreatic islets, a finding with potentially important implications for the progression of autoimmunity as well as for the establishment of transplant tolerance to this tissue.