Synapsins and neuroexocytosis: recent views from functional studies on synapsin null mutant mice.
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Animals↗
Biomedical subjects
O Cremona
Publications and source records attributed to O Cremona.
Dynamics of secretion.
In this short review, kinetic aspects of exocytosis are discussed. A special emphasis is put on recent data that highlight dynamic differences between neurotransmission and other forms of secretion.
Animals↗
Kinetics of neuronal and endocrine secretion.
Calcium (Ca2+) regulated secretion/exocytosis is a key mechanism for cell-cell communication. Neurotransmission and hormone release are the most studied and the best characterized of all secretion systems so far. Here, some dynamic aspects of secretory vesicle trafficking will be briefly reviewed with special emphasis on the differences between synaptic vesicle and dense-core vesicle turnover.
Animals↗
Synaptojanin 1 contributes to maintaining the stability of GABAergic transmission in primary cultures of cortical neurons.
Inhibitory synapses in the CNS can exhibit a considerable stability of neurotransmission over prolonged periods of high-frequency stimulation. Previously, we showed that synaptojanin 1 (SJ1), a presynaptic polyphosphoinositide phosphatase, is required for normal synaptic vesicle recycling (Cremona et al., 1999). We asked whether the stability of inhibitory synaptic responses was dependent on SJ1. Whole-cell patch-clamp recordings of unitary IPSCs were obtained in primary cortical cultures between cell pairs containing a presynaptic, fast-spiking inhibitory neuron (33.5-35 degrees C). Prolonged presynaptic stimulation (1000 stimuli, 2-20 Hz) evoked postsynaptic responses that decreased in size with a bi-exponential time course. A fast component developed within a few stimuli and was quantified with paired-pulse protocols. Paired-pulse depression (PPD) appeared to be independent of previous GABA release at intervals of >/=100 msec. The characteristics of PPD, and synaptic depression induced within the first approximately 80 stimuli in the trains, were unaltered in SJ1-deficient inhibitory synapses. A slow component of depression developed within hundreds of stimuli, and steady-state depression showed a sigmoidal dependence on stimulation frequency, with half-maximal depression at 6.0 +/- 0.5 Hz. Slow depression was increased when release probability was augmented, and there was a small negative correlation between consecutive synaptic amplitudes during steady-state depression, consistent with a presynaptic depletion process. Slow depression was increased in SJ1-deficient synapses, with half-maximal depression at 3.3 +/- 0.9 Hz, and the recovery was retarded approximately 3.6-fold. Our studies establish a link between a distinct kinetic component of physiologically monitored synaptic depression and a molecular modification known to affect synaptic vesicle reformation.
Action Potentials↗
Live stripping of clathrin-coated vesicles.
Vesicle budding requires recruitment of a coat, which must then be removed to allow fusion with the target compartment. In vitro assays have implicated Hsc70 and auxilin family members as key players in clathrin-coated vesicle uncoating. New in vivo studies now show that this is indeed the case and reveal additional functions of the Hsc70/auxilin complex.
Adaptor Proteins, Vesicular Transport↗
Phosphoinositides in membrane traffic at the synapse.
Inositol phospholipids represent a minor fraction of membrane phospholipids; yet they play important regulatory functions in signaling pathways and membrane traffic. The phosphorylated inositol ring can act either as a precursor for soluble intracellular messengers or as a binding site for cytosolic or membrane proteins. Hence, phosphorylation-dephosphorylation of phosphoinositides represents a mechanism for regulation of recruitment to the membrane of coat proteins, cytoskeletal scaffolds or signaling complexes and for the regulation of membrane proteins. Recent work suggests that phosphoinositide metabolism has an important role in membrane traffic at the synapse. PtdIns(4,5)P2 generation is implicated in the secretion of at least a subset of neurotransmitters. Furthermore, PtdIns(4,5)P2 plays a role in the nucleation of clathrin coats and of an actin-based cytoskeletal scaffold at endocytic zones of synapses, and PtdIns(4,5)P2 dephosphorylation accompanies the release of newly formed vesicles from these interactions. Thus, the reversible phosphorylation of inositol phospholipids may be one of the mechanisms governing the timing and vectorial progression of synaptic vesicle membranes during their exocytic-endocytic cycle.
Actins↗
Assignment of SYNJ1 to human chromosome 21q22.2 and Synj12 to the murine homologous region on chromosome 16C3-4 by in situ hybridization.
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Animals↗
Essential role of phosphoinositide metabolism in synaptic vesicle recycling.
Growing evidence suggests that phosphoinositides play an important role in membrane traffic. A polyphosphoinositide phosphatase, synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that synaptojanin 1-deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings. In cell-free assays, reduced phosphoinositide phosphatase activity correlated with increased association of clathrin coats with liposomes. Intracellular recording in hippocampal slices revealed enhanced synaptic depression during prolonged high-frequency stimulation followed by delayed recovery. These results provide genetic evidence for a crucial role of phosphoinositide metabolism in synaptic vesicle recycling.
Animals↗
The expression of alpha 2 beta 1 integrin and alpha smooth muscle actin in fibroblasts grown on collagen.
The maturation of connective tissue involves the organization of collagen fibres by resident fibroblasts. Fibroblast attachment to collagen has been demonstrated to involve cell surface receptors, integrins of the beta 1 family. Integrins are associated with cytoplasmic actin of microfilaments either directly or through focal adhesions. The major actin isoform of fibroblast microfilaments is beta actin and to a lesser extent alpha smooth muscle (alpha SM) actin. Cultured human dermal fibroblasts derived from adult dermis, newborn foreskin or keloid scar were grown on either uncoated or collagen-coated surfaces. The expression and synthesis of both alpha 2 beta 1 integrin and alpha SM actin were followed by immunohistology and immunoprecipitation. Fibroblasts on uncoated surfaces expressed little alpha 2 beta 1 integrin on their surface, while 20 per cent of them demonstrated alpha SM actin within microfilaments. Fibroblasts grown on a collagen-coated surface minimally expressed alpha SM actin in microfilament structures and a majority of the cells were positive for alpha 2 beta 1 integrin on their membranes. Using [35S]-methionine incorporation and immunoprecipitation, it was shown that fibroblasts grown in uncoated dishes synthesized more alpha SM actin than fibroblasts grown on collagen-coated dishes. In contrast, fibroblasts grown on collagen coated dishes synthesized more alpha 2 beta 1 integrin compared to the same cells grown on uncoated dishes. Fibroblasts maintained on a type I collagen upregulate the expression and synthesis of alpha 2 beta 1 integrin, and downregulate the expression and synthesis of alpha SM actin.
Actins↗
Expression of amphiphysin I, an autoantigen of paraneoplastic neurological syndromes, in breast cancer.
Amphiphysin I is a 128 kD protein highly concentrated in nerve terminals, where it has a putative role in endocytosis. It is a dominant autoantigen in patients with stiff-man syndrome associated with breast cancer, as well as in other paraneoplastic autoimmune neurological disorders. To elucidate the connection between amphiphysin I autoimmunity and cancer, we investigated its expression in breast cancer tissue. We report that amphiphysin I was expressed as two isoforms of 128 and 108 kD in the breast cancer of a patient with anti-amphiphysin I antibodies and paraneoplastic sensory neuronopathy. Amphiphysin I was also detectable at variable levels in several other human breast cancer tissues and cell lines and at low levels in normal mammary tissue and a variety of other non-neuronal tissues. The predominant amphiphysin I isoform expressed outside the brain in humans is the 108 kD isoform which represents an alternatively spliced variant of neuronal amphiphysin I missing a 42 amino acid insert. Our study suggests a link between amphiphysin I expression in cancer and amphiphysin I autoimmunity. The enhanced expression of amphiphysin I in some forms of cancer supports the hypothesis that amphiphysin family members may play a role in the biology of cancer cells.
Alternative Splicing↗
Amphiphysin II (SH3P9; BIN1), a member of the amphiphysin/Rvs family, is concentrated in the cortical cytomatrix of axon initial segments and nodes of ranvier in brain and around T tubules in skeletal muscle.
Amphiphysin (amphiphysin I), a dominant autoantigen in paraneoplastic Stiff-man syndrome, is a neuronal protein highly concentrated in nerve terminals, where it has a putative role in endocytosis. The yeast homologue of amphiphysin, Rvs167, has pleiotropic functions, including a role in endocytosis and in actin dynamics, suggesting that amphiphysin may also be implicated in the function of the presynaptic actin cytoskeleton. We report here the characterization of a second mammalian amphiphysin gene, amphiphysin II (SH3P9; BIN1), which encodes products primarily expressed in skeletal muscle and brain, as differentially spliced isoforms. In skeletal muscle, amphiphysin II is concentrated around T tubules, while in brain it is concentrated in the cytomatrix beneath the plasmamembrane of axon initial segments and nodes of Ranvier. In both these locations, amphiphysin II is colocalized with splice variants of ankyrin3 (ankyrinG), a component of the actin cytomatrix. In the same regions, the presence of clathrin has been reported. These findings support the hypothesis that, even in mammalian cells, amphiphysin/Rvs family members have a role both in endocytosis and in actin function and suggest that distinct amphiphysin isoforms contribute to define distinct domains of the cortical cytoplasm. Since amphiphysin II (BIN1) was reported to interact with Myc, it may also be implicated in a signaling pathway linking the cortical cytoplasm to nuclear function.
Adaptor Proteins, Signal Transducing↗
Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes.
The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA) but readily internalizes and degrades uPA:serpin complexes in a process that requires the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR-LRP). This process is accompanied by the internalization of uPAR which renders it resistant to phosphatidylinositol-specific phospholipase C (PI-PLC). In this paper we show that during internalization of uPA:serpins at 37 degrees C, analysed by FACScan, immunofluorescence and immunoelectron microscopy, an initial decrease of cell surface uPAR was observed, followed by its reappearance at later times. This effect was not due to redistribution of previously intracellular receptors, nor to the surface expression of newly synthesized uPAR. Recycling was directly demonstrated in cell surface-biotinylated, uPA:PAI-1-exposed cells in which biotinylated uPAR was first internalized and subsequently recycled back to the surface upon incubation at 37 degrees C. In fact, uPAR was resistant to PI-PLC after the 4 degrees C binding of uPA:PAI-1 to biotinylated cells, but upon incubation at 37 degrees C PI-PLC-sensitive biotinylated uPAR reappeared at the cell surface. Binding of uPA:PAI-1 by uPAR, while essential to initiate the whole process, was, however, dispensable at later stages as both internalization and recycling of uPAR could be observed also after dissociation of the bound ligand from the cell surface.
Animals↗
Synaptic vesicle endocytosis.
Exocytosis of synaptic vesicles is followed rapidly by reinternalization and recycling of their membranes. Recent studies have confirmed the key role of clathrin-mediated endocytosis in synaptic vesicle reformation and have identified new proteins that participate in this process. In addition, growing evidence suggests that lipids, primarily phosphoinositides, play an important role in synaptic vesicle recycling.
Animals↗
Changes in integrin and E-cadherin expression in neoplastic versus normal thyroid tissue.
UNLABELLED: BACK: The functional organization of polarized epithelia depends mostly on adhesion molecules belonging to the integrin and cadherin families. These molecules either recognize basement membrane components, such as laminins, or form intercellular junctions via homotypic interactions. Such tissue organization is often disrupted upon neoplastic transformation, and the resulting loss of functional polarization and cell cohesion might be a prerequisite for the invasive and metastatic behavior of carcinomas. PURPOSE: We studied modifications on thyroid adhesive mechanisms at various stages of neoplastic progression in terms of adhesion molecule expression, topography, and functional regulation by tyrosine kinases. Starting from this working hypothesis, we sought to identify one or more biological markers that would be suggestive of malignant transformation and poorer prognosis and that could be developed as a reliable indicator(s) in early diagnostic steps. METHODS: The study was carried out on both surgical samples and the corresponding fine-needle aspiration biopsy smears (numbers of specimens collected: 19 adenomas, seven follicular carcinomas, 13 papilary carcinomas, and 39 normal tissues). Immunohistochemistry of tissue sections and smears and immuno-precipitation and western blot analysis of protein extracts were done with a battery of monoclonal and polyclonal antibodies. Northern blotting was performed on RNA extracts from frozen tissue samples and use of an integrin subunit beta4 complementary DNA probe. RESULTS: Our findings can be summarized as follows: 1) In normal thyroid cells, the cooperative role of integrin alpha6beta4 and laminin 5/kalinin in hemidesmosome-mediated adhesion adhesion is missing, and recognition of the basal lamina occurs via integrin alpha3beta1 and laminin 1 and/or 2 (this pattern being maintained in adenomas but altered in carcinomas regardless of their histotype or differentiation grade); 2) only in carcinomas with clinical and/or histologic aggressiveness do neoexpression of integrin subunit beta4 and loss of laminin 2/merosin occur, indicating de novo assembly of integrin alpha6beta4; 3) pericellular redistribution and cytoskeletal disconnection of the E-cadherin-catenin complex occur; and 4) basal E-cadherin tyrosine phosphorylation decreases in carcinomas as compared with that in normal and adenomatous tissue. CONCLUSION: The malignant progression of thyroid tumors involves marked rearrangement of cell-basement membrane and cell-cell adhesion molecules and changes in their cytoskeleton linkage. These rearrangements are also easily and reproducibly detected on fine-needle aspiration biopsy smears. IMPLICATIONS: Immunodetection of adhesion molecules in sections and/or fine-needle smears may complement the toolbox of thyroid surgical pathologists; it may expand the possibilities of achieving a correct early diagnosis of thyroid tumors and of gaining some prognostic information on thyroid tumors.
Adenocarcinoma, Follicular↗
A family of transmembrane proteins with homology to the MET-hepatocyte growth factor receptor.
In hunting for unknown genes on the human X chromosome, we identified a cDNA in Xq28 encoding a transmembrane protein (SEX) of 1871 amino acids. SEX shares significant homology with the extracellular domain of the receptors encoded by the oncogenes MET, RON, and SEA [hepatocyte growth factor (HGF) receptor family]. Further screenings of cDNA libraries identified three additional sequences closely related to SEX: these were named SEP, OCT, and NOV and were located on human chromosomes 3p, 1, and 3q, respectively. The proteins encoded by these genes contain large cytoplasmic domains characterized by a distinctive highly conserved sequence (SEX domain). Northern blot analysis revealed different expression of the SEX family of genes in fetal tissues, with SEX, OCT, and NOV predominantly expressed in brain, and SEP expressed at highest levels in kidney. In situ hybridization analysis revealed that SEX has a distinctive pattern of expression in the developing nervous system of the mouse, where it is found in postmitotic neurons from the first stages of neuronal differentiation (9.5 day postcoitus). The SEX protein (220 kDa) is glycosylated and exposed at the cell surface. Unlike the receptors of the HGF family, p220SEX, a MET-SEX chimera or a constitutively dimerized TPR-SEX does not show tyrosine kinase activity. These data define a gene family (SEX family) involved in the development of neural and epithelial tissues, which encodes putative receptors with unexpected enzymatic or binding properties.
Amino Acid Sequence↗
Biosynthesis and apical localization of the urokinase receptor in polarized MDCK epithelial cells.
The biosynthesis and the surface localization of the urokinase plasminogen activator receptor (uPAR) were analysed in MDCK epithelial cells and in unpolarized fibroblasts. No differences were observed with respect to rate of synthesis, nature of precursors and time of surface appearance. uPAR was localized particularly at the focal and cell-cell contacts when expressed in fibroblasts. On the contrary, in MDCK cells uPAR was found mostly on the apical surface; in agreement with its localization, down-regulation of uPAR by the uPA-PAI-1 complex was observed only from the apical membrane.
3T3 Cells↗
The Met/HGF receptor is over-expressed in human osteosarcomas and is activated by either a paracrine or an autocrine circuit.
The c-MET oncogene encodes the receptor for the Hepatocyte Growth Factor/Scatter Factor (HGF), a cytokine that stimulates the invasive growth of normal and neoplastic cells. The Met/HGF receptor is expressed by epithelial cells and its ligand by cells of mesenchymal origin. Receptor-ligand interaction occurs via a paracrine circuit. We studied the expression of the Met/HGF receptor and of its ligand in mesenchymal human tumours by examining 39 clinical samples of bone tumours. The Met/HGF receptor was not detectable in the majority of bone tumours, as expected from their mesenchymal origin. Notably, the receptor was overexpressed in 60% of the osteosarcomas examined. In 12 osteosarcoma cell lines the Met/HGF receptor was overexpressed, phosphorylated by HGF stimulation and fully functional. HGF was detected in two out of seven clinical specimens of osteosarcoma. The ligand and the receptor are co-expressed in two clonal osteosarcoma cell lines. In these lines the Met/HGF receptor was constitutively phosphorylated; phosphorylation was suppressed by suramin treatment, a known blocker of autocrine loops. These data suggest that activation of the Met/HGF receptor by a paracrine or an autocrine mechanism might play a role in the particularly aggressive behaviour of osteosarcomas.
Bone Neoplasms↗
Differential protein expression in aortic smooth muscle cells cultured from newborn and aged rats.
Atherosclerosis is a complex disease in which smooth muscle cells (SMC) play a fundamental role. Work from several laboratories has suggested that in experimental models of atheromatosis SMC heterogeneity is important in the establishment of intimal thickening. Moreover it has been shown that SMC cultured from different situations in vivo maintain distinct phenotypic features in vitro. In order to find proteins differentially expressed in SMC cultured from newborn and aged rats, total protein extracts were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), high-resolution maps were built, and differentially expressed spots were identified by automatic computer analysis. Of the 14 differentially expressed protein spots, 4 were present in SMC of newborn and 10 in SMC of old animals; we describe their molecular weights and isoelectric points. One of these proteins (expressed only in cultured SMC of old rats) was successfully microsequenced for 16 amino acids and it was found identical to cellular retinol-binding protein. This results provides, to our knowledge, the first suggestion that retinoids are implicated in the differentiation and aging of vascular SMC.
Aging↗