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PubMed1999

Hsp47-dependent and -independent intracellular trafficking of type I collagen in corneal endothelial cells.

PURPOSE: Type I collagen is post-translationally regulated in corneal endothelial cells (CEC): CEC synthesize procollagen I and degrade it intracellularly. We investigated whether there is a Hsp47-independent pathway during intracellular trafficking of procollagen I. METHODS: Specific inhibitors were used to block intracellular transport of procollagen I and Hsp47. Immunocytochemical analysis was performed to determine the intracellular localization of the proteins of interest. RESULTS: When cells were treated with alpha,alpha'-dipyridyl, this specific inhibitor for collagen promoted retention in the endoplasmic reticulum (ER) of some of the underhydroxylated procollagen I, which was colocalized with Hsp47 in CEC. At the same time, another fraction of the alpha,alpha'-dipyridyl-induced underhydroxylated procollagen I was not located in the ER. When CEC were treated with brefeldin A, procollagen I and Hsp47 demonstrated a high degree of colocalization at the ER, whereas the inhibitor had less of an effect on the compartmentalization of procollagen I and prolyl 4-hydroxylase. When CEC were treated with either monensin or bafilomycin A1, procollagen I and Hsp47 were not colocalized: procollagen I was mostly localized at the Golgi area, while Hsp47 predominantly showed ER distribution. When colocalization of procollagen I and prolyl 4-hydroxylase was examined, a major population of procollagen I was not colocalized with prolyl 4-hydroxylase in the ER. CONCLUSIONS: These results indicate that some procollagen I and Hsp47 travel together from the ER to the cis-Golgi compartment and that a major population of procollagen I that may not be properly hydroxylated may be destroyed intracellularly via the Hsp47-independent pathway in CEC.

2,2'-Dipyridyl↗
PubMed2004

Tight junction protein MAGI-1 is up-regulated by transfection with connexin 32 in an immortalized mouse hepatic cell line: cDNA microarray analysis.

Gap junctions are considered to play a crucial role in differentiation of epithelial cells, including hepatocytes. Recently, we found that Cx32 but not Cx26 was closely related to tight junctional proteins in primary cultured rat hepatocytes (Kojima et al., Exp Cell Res 263:193-201, 2001) and that Cx32 formation and/or Cx32-mediated intercellular communication could induce expression and function of tight junctions in a mouse hepatic cell line (Kojima et al., Exp Cell Res 276:40-51, 2002). In this study, to investigate the mechanisms of induction of tight junctions by transfection with Cx32, we performed cDNA microarray analysis of Cx32 transfectants, compared with parental cells derived from Cx32-deficient hepatocytes. In cDNA microarray analysis, a 2.5-fold increase in expression of membrane-associated guanylate kinase with inverted orientation-1 (MAGI-1), which is known to be localized at adherens and tight junction regions, was observed. High expression of MAGI-1 in Cx32 transfectants was confirmed by Western blotting and RT-PCR. MAGI-1 was colocalized with occludin, claudin-2, ZO-1, and F-actin, but not with E-cadherin in the apical-most regions at cell borders of Cx32 transfectants, similar to junctional adhesion molecule-1 (JAM-1), which may play a crucial role in formation and assembly of tight junctions. Treatment with the gap junction blocker 18beta-glycyrrhetinic acid did not affect expression of MAGI-1 and JAM-1 in Cx32 transfectants. These results suggest that Cx32 expression is in part related to induction of tight junctions through modulation of MAGI-1 expression in an immortalized mouse hepatic cell line.

Actins↗
PubMed1997

CD137, a member of the tumor necrosis factor receptor family, is located on chromosome 1p36, in a cluster of related genes, and colocalizes with several malignancies.

CD137 (ILA/4-1BB) is a member of the tumor-necrosis-factor receptor family. Members of this receptor family and their structurally related ligands are important regulators of a wide variety of physiological processes and play an especially important role in the regulation of immune responses. CD137 regulates cell proliferation and survival of T-lymphocytes. Using Southern blot analysis and polymerase chain reaction, we localized the CD137 gene to chromosome 1p36. This chromosomal region harbors the genes of several other members of this receptor family and is associated with deletions and rearrangements in several malignancies.

Chromosome Deletion↗
PubMed2005

The role of SPECs, small Cdc42-binding proteins, in F-actin accumulation at the immunological synapse.

SPEC1 and SPEC2 are structurally similar Cdc42-binding proteins of 79 and 84 amino acid residues, respectively. We investigated the role of SPEC2 in T cell function due to its high mRNA expression in lymphocytes. Western blot analysis revealed abundant SPEC2 protein in lymphocytes, which in glutathione S-transferase-capture experiments specifically interacted with only GTP-bound Cdc42. Immunofluorescence experiments revealed that the SPEC2 protein was diffusely localized in the cytoplasm and at the cell membrane in unstimulated Jurkat T cells and Raji B cells. Recruitment of SPEC2 within Jurkat T cells to the antigen-presenting cell interface occurred following incubation with staphylococcal enterotoxin E superantigen-loaded B cells and colocalized there with F-actin and Cdc42. T cell receptor (TCR) activation studies using anti-CD3 antibody-coated polystyrene beads showed that SPEC2 was recruited to the site of bead contact, which was not observed with anti-major histocompatibility complex antibody-coated beads. Accumulation of SPEC2 following TCR engagement occurred as early as 5 min, before obvious F-actin accumulation. Biochemical studies with Jurkat T cells demonstrated that N-terminal cysteine residues in SPEC2 were palmitoylated. Overexpression studies of the related SPEC1 showed that it also was recruited to the activated TCR. Mutational analysis revealed that localization of SPEC1 to the TCR required two N-terminal cysteine residues. Furthermore, a SPEC1 Cdc42 Rac-interacting binding mutant, containing an intact N terminus but defective in Cdc42 binding, completely blocked F-actin accumulation at the activated TCR. Taken together these results suggest that SPECs may play important roles in Cdc42-mediated F-actin accumulation at the immunological synapse.

Actins↗
PubMed2006

Expression of fractalkine and fractalkine receptor in urinary bladder after cyclophosphamide (CYP)-induced cystitis.

Alterations in the expression of the chemokine, fractalkine (CX3CL1), were examined in the urinary bladder after cyclophosphamide (CYP)-induced cystitis of varying duration: acute (4 h or 48 h), or chronic (10 day). CYP-induced cystitis significantly (p<or=0.01) increased fractalkine protein expression in the urinary bladder with acute (48 h) and chronic CYP treatment. Western blot analysis also demonstrated significantly (p<or=0.01) increased fractalkine expression in the whole urinary bladder with acute (1.5-2.2-fold) and chronic (3-fold) CYP-induced cystitis. Immunohistochemistry for fractalkine-immunoreactivity revealed little fractalkine-IR in control or acute (4 h) CYP-treated rat urinary bladders except in a vascular bed but showed no colocalization with nerve fibers in the suburothelial plexus in any experimental group. However, expression was significantly (p<or=0.001) upregulated in the urothelium with 48 h or chronic CYP treatment. Similarly, fractalkine receptor (CX3CR1)-IR was significantly (p<or=0.001) upregulated in the urothelium with 48 h or chronic CYP treatment. These studies demonstrated upregulation of the chemokine, fractalkine, in the urinary bladder and specifically in the urothelium with CYP-induced cystitis. Chemokines, and specifically, fractalkine, may be another class of neuromodulatory agents upregulated in the urinary bladder that can affect micturition function and sensory processing with cystitis and may represent novel, drug targets for cystitis.

Analysis of Variance↗
PubMed1991

Disruption of the Golgi apparatus with brefeldin A does not destabilize the associated detyrosinated microtubule network.

Stable subsets of microtubules (MTs) are often enriched in detyrosinated alpha-tubulin. Recently it has been found that the Golgi apparatus is associated with a subset of relatively stable MTs and that detyrosinated MTs colocalize spatially and temporally with the Golgi apparatus in several cell lines. To determine whether the Golgi apparatus actively stabilizes associated MTs and thus allows their time-dependent detyrosination, we have used the drug brefeldin A (BFA) to disrupt the Golgi apparatus and have monitored changes in the Golgi apparatus and MT populations using simultaneous immunofluorescence and fluorescent lectin microscopy. We found that although BFA caused the Golgi apparatus to completely redistribute to the endoplasmic reticulum (ER), the detyrosinated MTs were not disrupted and remained in a juxtanuclear region. By Western blot analysis we found that even after 6 h of continuous exposure of cells to BFA, there was no detectable reduction in the level of detyrosinated alpha-tubulin. Simultaneous treatment with nocodazole and BFA led to a complete disruption of all MTs and normal Golgi structure/organization. Upon removal of nocodazole in the continued presence of BFA, we found that the detyrosinated MTs reformed in a compact juxtanuclear location in the absence of an intact Golgi complex. Finally, we found that the detyrosinated MTs colocalized precisely with a BFA-resistant structure that binds to the lectin, wheat germ agglutinin. We conclude that the juxtanuclear detyrosinated MTs are not actively stabilized by association with BFA-sensitive Golgi membranes. However, another closely associated structure which binds wheat germ agglutinin may serve to stabilize the juxtanuclear MTs. Alternatively, the MT organizing center (MTOC) and/or MT-associated proteins (MAPs) may organize and stabilize the juxtanuclear detyrosinated MTs.

Animals↗
PubMed2000

Localization of a syntaxin isoform, syntaxin 2, to the acrosomal region of rodent spermatozoa.

The acrosome reaction includes a membrane fusion event that is a prerequisite for sperm penetration through the zona pellucida and subsequent fertilization. Since SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins have been shown to be key players in membrane fusion during regulated exocytosis in nerve terminals and secretory cells, and since the acrosome reaction has some features in common with regulated exocytosis, we hypothesized that SNARE proteins might also regulate acrosomal exocytosis. RT-PCR analysis demonstrated the expression of SNARE proteins, three isoforms of syntaxin 2 (2A, 2B, and 2C) and syntaxin 4A, in rat testes. Immunoblot analysis with anti-syntaxin 2 antibody showed that the protein was expressed in rodent spermatozoa, and that it was associated with membrane components of spermatozoa prepared by sucrose density gradient centrifugation. Confocal laser scanning microscopy with double immunolabeling revealed that syntaxin 2 was colocalized with acrin 1, a 90 kDa acrosomal protein, over the acrosomal region of spermatozoa but was not associated with the posterior half of head or tail. Localization of syntaxin 2 over the acrosomal region was supported by the finding that it was shed from sperm heads during an acrosome reaction induced by calcium ionophore A23187 in vitro. In view of the putative role of syntaxin proteins in other membrane fusion systems, these data suggest that syntaxin 2 may be involved in regulating the acrosomal reaction in rodent spermatozoa.

Acrosome↗
PubMed2004

Chemokine receptors are expressed widely by embryonic and adult neural progenitor cells.

We investigated the expression and functions of chemokine receptors in neural progenitor cells isolated from embryonic and adult mice. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated mRNA expression for most known chemokine receptors in neural progenitor cells grown as neurospheres from embryonic (E17) and adult (4-week-old) mice. The expression of CXCR4 receptors was demonstrated further in E17 neurospheres using immunohistochemistry, in situ hybridization, Northern blot analysis and fura-2-based Ca(2+) imaging. Most neurospheres grown from E17 mice responded to stromal cell-derived factor-1 (SDF-1/CXCL12) in Ca(2+) imaging studies. In addition, immunohistochemical studies demonstrated that these neurospheres consisted of dividing cells that uniformly colocalized nestin and CXCR4 receptors. Differentiation of E17 neurospheres yielded astrocytes and neurons exhibiting several different phenotypes, including expression of calbindin, calretinin, gamma-aminobutyric acid (GABA), and glutamate, and many also coexpressed CXCR4 receptors. In addition, neurospheres grown from the subventricular zone (SVZ) of 4-week-old mice exhibited large increases in Ca(2+) in response to CXCL12 and several other chemokines. In comparison, neurospheres prepared from olfactory bulb of adult mice exhibited only small Ca(2+) responses to CXCL12, whereas neurospheres prepared from hippocampus were insensitive to CXCL12, although they did respond to other chemokines. Investigations designed to investigate whether CXCL12 can act as a chemoattractant demonstrated that cells dissociated from E17 or adult SVZ neurospheres migrated toward an CXCL12 gradient and this was blocked by the CXCR4 antagonist AMD3100. These results illustrate widespread chemokine sensitivity of embryonic and adult neural progenitor cells and support the view that chemokines may be of general importance in control of progenitor cell migration in embryonic and adult brain.

Animals↗
PubMed1997

Fibronectin distribution in human bone marrow stroma: matrix assembly and tumor cell adhesion via alpha5 beta1 integrin.

Tumor cell interactions with fibronectin (FN) are important for the development of secondary tumors inside the bone marrow stroma. We studied and compared the in situ distribution of FN in paraffin-embedded human bone marrow sections and investigated the in vitro regulation of FN assemblage by bone marrow stromal cells (BMSC). Finally, the role of FN in the interaction of BMSC with tumor cells was studied. Fine elongated FN-positive cell extensions, probably of stromal cell origin, were observed as well as a limited amount of extracellular FN deposits in connective tissues around capillaries and sinusoids. In vitro studies, using the confocal laser scanning microscope, showed that BMSC produced a high amount of FN with a characteristic extracellular matrix formation in an extensive network. FN matrix formation was predominantly detected at contact sites between cultured BMSC. In in vitro cultures with low cell concentrations and in vivo with a limited number of stromal cell contacts only limited matrix was found. From previous studies it is known that the alpha5 beta1 integrin is involved in the regulation of FN assembly. Here the role of the alpha5-subunit of this integrin was investigated. By using two different monoclonal antibodies (mAb) against the alpha5-subunit (2H6 and mAb16) the assembly of endogenous FN was completely blocked, indicating that these antibodies are directed against the active epitope. Another mAb (mAb11) against the alpha5-subunit did not affect the FN assemblage. Codistribution analysis of alpha5-subunits, alpha v-subunits, actin, and FN demonstrated that the alpha5 beta1 integrin is associated with FN and not with intracellular actin. Integrins alpha(v) beta1, alpha(v) beta3, and alpha(v) beta5, also ligands of FN, did not colocalize with FN. Codistribution of alpha v with the terminal ends of actin and not with FN indicates that alpha(v)-subunits are mainly directed to vitronectin rather than to FN. The dominant role of alpha5 beta1 in FN interaction is underlined by effective blocking of tumor cell adhesion with BMSC using anti-alpha5, anti-beta1, and anti-FN antibodies. These results emphasize the important role of alpha5 integrin subunit in FN matrix assembly in human BMSC and an exclusive role of alpha5 beta1 in the anchorage and regulation of FN-mediated adhesion processes in the bone marrow.

Actins↗
PubMed2005

The RAGE pathway in inflammatory myopathies and limb girdle muscular dystrophy.

Oxidative stress and nuclear factor-kappaB (NF-kappaB) activation are linked to the pathogenesis of many metabolic, degenerative, and chronic inflammatory diseases. Activation of the receptor for advanced glycation end products (RAGE) by its specific ligand N(epsilon)-carboxymethyllysine (CML) results in the activation of NF-kappaB and the production of proinflammatory cytokines. To determine whether engagement of RAGE contributes to the pathogenesis of inflammatory myopathies, we performed immunohistochemical studies on the presence of CML-modified proteins, RAGE and activated NF-kappaB in muscle biopsies of patients with polymyositis (PM, n=10), dermatomyositis (DM, n=10), limb girdle muscular dystrophy (LGMD, n=10) and in 10 controls with normal muscle biopsy results. In inflammatory myopathies CML, RAGE and NF-kappaB were detected in mononuclear cells and in regenerating muscle fibers. CML, NF-kappaB and, to a lesser extent, RAGE were also found in degenerating muscle fibers, but colocalization of CML, RAGE and NF-kappaB was only seen in infiltrating mononuclear cells and regenerating muscle fibers. Immunofluorescence double labeling demonstrated an expression of CML, RAGE and NF-kappaB in CD4-, CD8-, CD22- and CD68-positive mononuclear cells. Western blot analysis showed an increased immunoreactivity for CML-modified proteins in PM and DM. In LGMD, CML, RAGE and NF-kappaB were found in regenerating muscle fibers and less frequently in degenerating muscle fibers, and with lower staining intensities than in inflammatory myopathies. Our data suggests that the CML-RAGE-NF-kappaB pathway is an evident proinflammatory pathomechanism in mononuclear effector cells in PM and DM. RAGE-mediated NF-kappaB activation may be involved in muscle fiber regeneration in inflammatory myopathies and LGMD.

Adult↗
PubMed1992

Adenovirus capsid proteins interact with HSP70 proteins after penetration in human or rodent cells.

Soon after penetration of adenovirus serotype 2 in BHK-21 and HeLa cells, HSP70 and HSC70 proteins become associated with the viral capsid. By analysis with a polyclonal antibody derived from a fusion protein containing the C-terminal domain, 290 amino acids of HSP70, and using both immunological methods and infected cells fractionation we observed that a significant amount of HSP70 proteins moved to the nucleus and colocalized with the adenovirus particles. HSP70 proteins of infected cells were isolated as a complex cross-linked with intracytoplasmic adenovirus type 2. By coprecipitation, using a polyclonal-specific antiserum derived from the fusion protein, or two different monoclonal-specific antisera, we showed that HSP70 and HSC70 proteins were associated with hexon, the major adenovirus capsid protein.

Adenoviridae↗
PubMed2026

Plasma proteins are integral to cross-tissue gene regulatory networks implicated in cardiometabolic disorders and coronary artery disease.

The plasma proteome has demonstrated promise for identifying diagnostic markers for cardiometabolic disorders (CMDs) and coronary artery disease (CAD). However, identifying the organ of origin for these biomarkers is critical for establishing biological relevance. We performed a multi-omic integrative analysis across multiple tissues from the STARNET study by profiling 974 plasma proteins in 532 CAD patients, integrating RNA sequencing (RNA-seq) data from the arterial wall, major metabolic organs, and blood. We identified 144 cis-protein quantitative trait loci in plasma, colocalizing with tissue cis-expression quantitative trait loci. Additionally, by mapping tissue mRNA "seed genes," we traced 262 plasma proteins to their source organs, primarily the liver. Crucially, we found that 851 plasma proteins are associated with the activity of cross-tissue gene regulatory networks (GRNs), including GRNs implicated in CMD and CAD development. Our findings demonstrate that plasma proteins are integral components of GRNs, with potential for developing reliable diagnostics and precise therapeutic targets. A record of this paper's transparent peer review process is included in the supplemental information.

cardiometabolic disorders↗
PubMed2006

NFATc1 expression in the developing heart valves is responsive to the RANKL pathway and is required for endocardial expression of cathepsin K.

NFATc1 is necessary for remodeling endocardial cushions into mature heart valve leaflets and is also an essential effector of receptor activator of NFkappaB ligand (RANKL) signaling required for transcriptional activation of bone matrix remodeling enzymes during osteoclast differentiation. Therefore, developing heart valves were examined to determine if NFATc1 functions in the RANKL pathway during leaflet remodeling. Key components of RANKL signal transduction including RANKL, its receptor RANK, and the downstream remodeling enzyme cathepsin K (Ctsk) are expressed in the heart during valve remodeling and colocalize with NFATc1 in developing valve endocardium. However, the absence of tartrate-resistant acid phosphatase (TRAP) activity and the lack of F4/80-positive macrophage lineage contribution to the remodeling valves demonstrate that certain aspects of osteoclast RANKL function are not shared during valve formation. Analysis of NFATc1-/- mouse embryos shows that NFATc1 is specifically required for endocardial expression of RANKL and Ctsk during valve formation. In addition, RANKL treatment augments expression of NFATc1 and Ctsk in embryonic heart cultures, and the RANKL-mediated increase in Ctsk expression is dependent on NFATc1. Together, these results support a role for RANKL signaling during heart valve development and suggest that valve leaflet morphogenesis involves NFATc1-dependent expression of remodeling enzymes including Ctsk.

Animals↗
PubMed2002

The melanocytic protein Melan-A/MART-1 has a subcellular localization distinct from typical melanosomal proteins.

To delineate the role of the melanocyte lineage-specific protein Melan-A/MART-1 in melanogenic functions, a set of biochemical and microscopical studies was performed. Biochemical analysis revealed that Melan-A/MART-1 is post-translationally acylated and undergoes a rapid turnover in a pigmented melanoma cell line. Immunofluorescence and immunoelectron microscopy analyses indicated that Melan-A/MART-1 is mainly located in the Golgi area and only partially colocalizes with melanosomal proteins. Quantitative immunoelectron microscopy showed that the highest proportion of the cellular content of Melan-A/MART-1 was found in small vesicles and tubules throughout the cell, whereas the concentration was maximal in the Golgi region, particularly the trans-Golgi network. Substantial labeling was also present on melanosomes, endosomes, ER, nuclear envelope, and plasma membrane. In early endosomes, Melan-A was enriched in areas of the limiting membrane covered by a bi-layered coat, a structural characteristic of melanosomal precursor compartments. Upon melanosome maturation, Melan-A concentration decreased and its predominant localization shifted from the limiting membrane to internal vesicle membranes. In conjunction with its acylation, the high expression levels of Melan-A in the trans-Golgi network, in dispersed vesicles, and on the limiting membrane of premelanosomes indicate that the protein may play a role during the early stage of melanosome biogenesis.

Antigens, Neoplasm↗
PubMed2003

Dissociation of DNA polymerase alpha-primase complex during meiosis in Coprinus cinereus.

Previously, the activity of DNA polymerase alpha was found in the meiotic prophase I including non-S phase stages, in the basidiomycetes, Coprinus cinereus. To study DNA polymerase alpha during meiosis, we cloned cDNAs for the C. cinereus DNA polymerase alpha catalytic subunit (p140) and C. cinereus primase small subunit (p48). Northern analysis indicated that both p140 and p48 are expressed not only at S phase but also during the leptotene/zygotene stages of meiotic prophase I. In situ immuno-staining of cells at meiotic prophase I revealed a sub population of p48 that does not colocalize with p140 in nuclei. We also purified the pol alpha-primase complex from meiotic cells by column chromatography and characterized its biochemical properties. We found a subpopulation of primase that was separated from the pol alpha-primase complex by phosphocellulose column chromatography. Glycerol gradient density sedimentation results indicated that the amount of intact pol alpha-primase complex in crude extract is reduced, and that a smaller complex appears upon meiotic development. These results suggest that the form of the DNA polymerase alpha-primase complex is altered during meiotic development.

Blotting, Northern↗
PubMed2001

Altered extracellular signal-regulated kinase signal transduction by the muscarinic acetylcholine and metabotropic glutamate receptors after cerebral ischemia.

To determine whether muscarinic acetylcholine receptors (mAChR) in the post-ischemic hippocampus may be involved in altered extracellular signal-regulated kinases (ERK) signal transduction, we have investigated changes in the activity of ERK1/2 induced by a muscarinic agonist, carbachol. Cerebral ischemia was produced in the rat by injecting 900 microspheres (48 microm in diameter) into the right internal carotid artery. Applying carbachol to the contralateral hippocampal slices from ischemic rats increased the phosphorylation of ERK1/2 but did not increase phosphorylation in the ipsilateral hippocampus. Analysis of M(1) mAChR binding showed that there was no significant difference in the number and K(d) values between the hippocampi from naïve and ischemic rats. Immunoblotting analysis showed no significant difference in the amount of M(1) mAChR in both hemispheres. In contrast to carbachol stimulation, the protein kinase C activator induced an activation of ERK1/2 in the ipsilateral hippocampus. This increase was shown to occur in neurons by immunofluorescence colocalization study. Carbachol-stimulated tyrosine phosphorylation of the G alpha(q/11), inositol 1,4,5-trisphosphate formation, and association of G alpha(q/11) with phospholipase C beta 1 were attenuated in the ipsilateral hippocampus. We also found that stimulation of group I metabotropic glutamate receptors, which are linked to G alpha(q/11), failed to increase in phosphorylation of ERK1/2 in the ipsilateral hippocampus. These results suggest that failure in receptor-mediated tyrosine phosphorylation of the G alpha(q/11) subunit and a defect in receptor-G alpha(q/11-)effector coupling in the ischemic hippocampus may be involved in alterations of ERK signal transduction.

Animals↗
PubMed2006

A repeated IMP-binding motif controls oskar mRNA translation and anchoring independently of Drosophila melanogaster IMP.

Zip code-binding protein 1 (ZBP-1) and its Xenopus laevis homologue, Vg1 RNA and endoplasmic reticulum-associated protein (VERA)/Vg1 RNA-binding protein (RBP), bind repeated motifs in the 3' untranslated regions (UTRs) of localized mRNAs. Although these motifs are required for RNA localization, the necessity of ZBP-1/VERA remains unresolved. We address the role of ZBP-1/VERA through analysis of the Drosophila melanogaster homologue insulin growth factor II mRNA-binding protein (IMP). Using systematic evolution of ligands by exponential enrichment, we identified the IMP-binding element (IBE) UUUAY, a motif that occurs 13 times in the oskar 3'UTR. IMP colocalizes with oskar mRNA at the oocyte posterior, and this depends on the IBEs. Furthermore, mutation of all, or subsets of, the IBEs prevents oskar mRNA translation and anchoring at the posterior. However, oocytes lacking IMP localize and translate oskar mRNA normally, illustrating that one cannot necessarily infer the function of an RBP from mutations in its binding sites. Thus, the translational activation of oskar mRNA must depend on the binding of another factor to the IBEs, and IMP may serve a different purpose, such as masking IBEs in RNAs where they occur by chance. Our findings establish a parallel requirement for IBEs in the regulation of localized maternal mRNAs in D. melanogaster and X. laevis.

3' Untranslated Regions↗
PubMed2003

Phosphoinositides, ezrin/moesin, and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors.

The post-Golgi trafficking of rhodopsin in photoreceptor cells is mediated by rhodopsin-bearing transport carriers (RTCs) and regulated by the small GTPase rab8. In this work, we took a combined pharmacological-proteomic approach to uncover new regulators of RTC trafficking toward the specialized light-sensitive organelle, the rod outer segment (ROS). We perturbed phospholipid synthesis by activating phospholipase D with sphingosine 1-phosphate (S1P) or inhibiting phosphatidic acid phosphohydrolase by propranolol (Ppl). S1P stimulated the overall rate of membrane trafficking toward the ROS. Ppl stimulated budding of RTCs, but blocked membrane delivery to the ROS. Ppl caused accumulation of RTCs in the vicinity of the fusion sites, suggesting a defect in tethering, similar to the previously described phenotype of the rab8T22N mutant. Proteomic analysis of RTCs accumulated upon Ppl treatment showed a significant decrease in phosphatidylinositol-4,5-bisphosphate-binding proteins ezrin and/or moesin. Ppl induced redistribution of moesin, actin and the small GTPase rac1 from RTCs into the cytosol. By confocal microscopy, ezrin/moesin and rac1 colocalized with rab8 on RTCs at the sites of their fusion with the plasma membrane; however, this distribution was lost upon Ppl treatment. Our data suggest that in photoreceptors phosphatidylinositol-4,5-bisphosphate, moesin, actin, and rac1 act in concert with rab8 to regulate tethering and fusion of RTCs. Consequentially, they are necessary for rhodopsin-laden membrane delivery to the ROS, thus controlling the critical steps in the biogenesis of the light-detecting organelle.

Actins↗