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1.
Glia ; 71(10): 2456-2472, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37395323

RESUMO

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder in which patients lose motor functions due to progressive loss of motor neurons in the cortex, brainstem, and spinal cord. Whilst the loss of neurons is central to the disease, it is becoming clear that glia, specifically astrocytes, contribute to the onset and progression of neurodegeneration. Astrocytes play an important role in maintaining ion homeostasis in the extracellular milieu and regulate multiple brain functions by altering their extracellular concentrations. In this study, we have investigated the ability of astrocytes to maintain K+ homeostasis in the brain via direct measurement of the astrocytic K+ clearance rate in the motor and somatosensory cortices of an ALS mouse model (SOD1G93A ). Using electrophysiological recordings from acute brain slices, we show region-specific alterations in the K+ clearance rate, which was significantly reduced in the primary motor cortex but not the somatosensory cortex. This decrease was accompanied by significant changes in astrocytic morphology, impaired conductivity via Kir4.1 channels and low coupling ratio in astrocytic networks in the motor cortex, which affected their ability to form the K+ gradient needed to disperse K+ through the astrocytic syncytium. These findings indicate that the supportive function astrocytes typically provide to motoneurons is diminished during disease progression and provides a potential explanation for the increased vulnerability of motoneurons in ALS.


Assuntos
Esclerose Lateral Amiotrófica , Camundongos , Animais , Astrócitos , Superóxido Dismutase-1 , Neurônios Motores/fisiologia , Medula Espinal , Modelos Animais de Doenças , Progressão da Doença , Camundongos Transgênicos , Superóxido Dismutase
2.
Cerebellum ; 21(3): 404-424, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34324160

RESUMO

GFAP-IL6 transgenic mice are characterised by astroglial and microglial activation predominantly in the cerebellum, hallmarks of many neuroinflammatory conditions. However, information available regarding the proteome profile associated with IL-6 overexpression in the mouse brain is limited. This study investigated the cerebellum proteome using a top-down proteomics approach using 2-dimensional gel electrophoresis followed by liquid chromatography-coupled tandem mass spectrometry and correlated these data with motor deficits using the elevated beam walking and accelerod tests. In a detailed proteomic analysis, a total of 67 differentially expressed proteoforms including 47 cytosolic and 20 membrane-bound proteoforms were identified. Bioinformatics and literature mining analyses revealed that these proteins were associated with three distinct classes: metabolic and neurodegenerative processes as well as protein aggregation. The GFAP-IL6 mice exhibited impaired motor skills in the elevated beam walking test measured by their average scores of 'number of footslips' and 'time to traverse' values. Correlation of the proteoforms' expression levels with the motor test scores showed a significant positive correlation to peroxiredoxin-6 and negative correlation to alpha-internexin and mitochondrial cristae subunit Mic19. These findings suggest that the observed changes in the proteoform levels caused by IL-6 overexpression might contribute to the motor function deficits.


Assuntos
Proteoma , Proteômica , Animais , Interleucina-6 , Camundongos , Doenças Neuroinflamatórias , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos
3.
Traffic ; 16(11): 1174-92, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26399547

RESUMO

Cytokinesis is the final stage of cell division and produces two independent daughter cells. Vesicles derived from internal membrane stores, such as the Golgi, lysosomes, and early and recycling endosomes accumulate at the intracellular bridge (ICB) during cytokinesis. Here, we use electron tomography to show that many ICB vesicles are not independent but connected, forming a newly described ICB vesicular structure - narrow tubules that are often branched. These 'midbody tubules' labelled with horseradish peroxidase (HRP) within 10 min after addition to the surrounding medium demonstrating that they are derived from endocytosis. HRP-labelled vesicles and tubules were observed at the rim of the ICB after only 1 min, suggesting that midbody tubules are likely to be generated by local endocytosis occurring at the ICB rim. Indeed, at least one tubule was open to the extracellular space, indicative of a local origin within the ICB. Inhibition of cholesterol-dependent endocytosis by exposure to methyl-ß-cyclodextrin and filipin reduced formation of HRP-labelled midbody tubules, and induced multinucleation following ICB formation. In contrast, dynamin inhibitors, which block clathrin-mediated endocytosis, induced multinucleation but had no effect on the formation of HRP-labelled midbody tubules. Therefore, our data reveal the existence of a cholesterol-dependent endocytic pathway occurring locally at the ICB, which contributes to the accumulation of vesicles and tubules that contribute to the completion of cytokinesis.


Assuntos
Colesterol/metabolismo , Citocinese/fisiologia , Endocitose/fisiologia , Endossomos/metabolismo , Lisossomos/metabolismo , Complexo de Golgi/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Microscopia Eletrônica/métodos , beta-Ciclodextrinas/metabolismo
4.
Proteomics ; 14(7-8): 872-89, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24452924

RESUMO

The large-scale resolution and detection of proteins from complex native mixtures is fundamental to quantitative proteomic analyses. Comprehensive analyses depend on careful tissue handling and quantitative protein extraction and assessment. To most effectively link these analyses with an understanding of underlying molecular mechanisms, it is critical that all protein types - isoforms, splice variants and those with functionally important PTMs - are quantitatively extracted with high reproducibility. Methodological details concerning protein extraction and resolution using 2DE are discussed with reference to current in-gel protein detection limits. We confirm a significant increase in total protein, and establish that extraction, resolution and detection of phospho- and glycoproteins are improved following automated frozen disruption relative to manual homogenisation. The quality of 2DE protein resolution is established using third-dimension separations and 'deep imaging'; substantially more proteins/protein species than previously realised are actually resolved by 2DE. Thus, the key issue for effective proteome analyses is most likely to be detection, not resolution. Thus, these systematic methodological and technical advances further solidify the role of 2DE in top-down proteomics. By routinely assessing as much proteomic data from a sample as possible, 2DE enables more detailed and critical insights into molecular mechanisms underlying different physiological states.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteínas/isolamento & purificação , Proteômica , Misturas Complexas , Proteínas/classificação , Proteínas/metabolismo
5.
J Oncol ; 2024: 9925970, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38249992

RESUMO

Zinc dyshomeostasis is manifested in breast and prostate cancer cells. This study attempted to uncover the molecular details prodded by the change of extracellular zinc by employing a panel of normal and cancerous breast and prostate cell lines coupled with the top-down proteomics with two-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry. The protein samples were generated from MCF-7 breast cancer cells, MCF10A normal breast cells, PC3 prostate cancer cells, and RWPE-1 normal prostate cells with or without exogenous zinc exposure in a time course (T0 and T120). By comparing the cancer cells vs respective normal epithelial cells without zinc treatment (T0), differentially expressed proteins (23 upregulated and 18 downregulated in MCF-7 cells; 14 upregulated and 30 downregulated in PC3 cells) were identified, which provides insights into the intrinsic differences of breast and prostate cancer cells. The dynamic protein landscapes in the cancer cells prodded by the extracellular zinc treatment reveal the potential roles of the identified zinc-responsive proteins (e.g., triosephosphate isomerase, S100A13, tumour proteins hD53 and hD54, and tumour suppressor prohibitin) in breast and prostate cancers. This study, for the first time, simultaneously investigated the two kinds of cancer cells related to zinc dyshomeostasis, and the findings shed light on the molecular understanding of the breast and prostate cancer cells in response to extracellular zinc variation.

6.
J Trace Elem Med Biol ; 86: 127500, 2024 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-39047373

RESUMO

BACKGROUND: Zinc transport proteins (ZIP and ZnT), metallothioneins (MT) and protein kinase CK2 are involved in dysregulation of zinc homeostasis in breast and prostate cancer cells. Following up our previous research, we targeted ZIP12, ZnT1, MT2A and CK2 in this study by investigating their expression levels and protein localisation. METHODS: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence confocal microscopy were employed to quantify the expression of ZIP12, ZnT1, MT2A and CK2 subunits in a panel of breast and prostate cell lines without or with extracellular zinc exposure. The cellular localisations of these target proteins were also examined by immunofluorescence confocal microscopy. RESULTS: In response to the extracellular zinc exposure, the gene expression was elevated for SLC39A12 (ZIP12), SLC30A1 (ZnT1) and MT2A (MT2A) in normal prostate epithelial cells (RWPE-1) in contrast to their cancerous counterparts (PC3 and DU145), whilst the gene expression was higher for SLC39A12 (ZIP12) and SLC30A1 (ZnT1) in both normal (MCF10A) and basal breast cancer cells (MDA-MB-231) compared to luminal breast cancer cells (MCF-7). At the protein level, the expression for both ZIP12 and ZnT1 was trending lower in the time course for the breast cancer cells whilst their expression was remained constant in the normal breast epithelial cells. The expression of ZIP12 in prostate cancer cells was higher than the normal prostate cells. The protein expression for CK2 α/αꞌ and CK2ß was markedly higher in prostate cancer cells than the normal prostate cells. Upon extracellular zinc exposure, ZIP12 was, for the first time, conspicuously localised in the plasma membrane of breast cancer cells but not in normal breast epithelial cells and prostate cells. ZnT1 is only localised in the plasma membrane of breast cancer cells. MT2A is distinctively seen close to the plasma membrane in breast cancer cells. CK2 is also for the first time shown to be localised in proximity to the plasma membrane of breast cancer cells. CONCLUSION: The findings, particularly the localisation of ZIP12 and CK2, are novel and significant for our understanding of zinc homeostasis in breast and prostate cancer cells.

7.
Biochim Biophys Acta ; 1813(10): 1689-99, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21195118

RESUMO

Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO(2) chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and Glutathione-S-Transferase-tagged-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.


Assuntos
Proteína Quinase CDC2/metabolismo , Citocinese , Dinamina II/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Animais , Proteína Quinase CDC2/fisiologia , Domínio Catalítico , Células Cultivadas , Ciclina B1/metabolismo , Ciclina B1/fisiologia , Citocinese/genética , Citocinese/fisiologia , Dinamina II/química , Dinamina II/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Fosforilação/genética , Ratos , Serina/genética , Ovinos , Spodoptera
8.
Metallomics ; 14(8)2022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35657662

RESUMO

Zn2+ dyshomeostasis is an intriguing phenomenon in breast and prostate cancers, with breast cancer cells exhibiting higher intracellular Zn2+ level compared to their corresponding normal epithelial cells, in contrast to the low Zn2+ level in prostate cancer cells. In order to gain molecular insights into the zinc homeostasis of breast and prostate cancer cells, this study profiled the expression of 28 genes, including 14 zinc importer genes (SLC39A1-14) that encode Zrt/Irt-like proteins 1-14 to transport Zn2+ into the cytoplasm, 10 zinc exporter genes (SLC30A1-10) which encode Zn2+ transporters 1-10 to transport Zn2+ out of the cytoplasm, and 4 metallothionein genes (MT1B, MT1F, MT1X, MT2A) in breast (MCF10A, MCF-7, MDA-MB-231) and prostate (RWPE-1, PC3, DU145) cell lines in response to extracellular zinc exposures at a mild cytotoxic dosage and a benign dosage. The RNA samples were prepared at 0 min (T0), 30 min (T30), and 120 min (T120) in a time course with or without zinc exposure, which were used for profiling the baseline and dynamic gene expression. The up-regulation of MT genes was observed across the breast and prostate cancer cell lines. The expression landscape of SLC39A and SLC30A was revealed by the quantitative reverse transcription polymerase chain reaction data of this study, which sheds light on the divergence of intracellular Zn2+ levels for breast and prostate cancer cells. Taken together, the findings are valuable in unraveling the molecular intricacy of zinc homeostasis in breast and prostate cancer cells.


Assuntos
Neoplasias da Mama , Neoplasias da Próstata , Neoplasias da Mama/metabolismo , Homeostase , Humanos , Masculino , Metalotioneína/metabolismo , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Zinco/metabolismo
9.
Nat Cell Biol ; 5(8): 701-10, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12855954

RESUMO

Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin I on Ser 774 and Ser 778 in vitro, which are identical to its endogenous phosphorylation sites in vivo. Cdk5 antagonists and expression of dominant-negative Cdk5 block phosphorylation of dynamin I, but not of amphiphysin or AP180, in nerve terminals and inhibit SVE. Thus Cdk5 has an essential role in SVE and is the first dephosphin kinase identified in nerve terminals.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Endocitose/fisiologia , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Motivos de Aminoácidos , Animais , Quinase 5 Dependente de Ciclina , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Dinamina I/genética , Dinamina I/metabolismo , Inibidores Enzimáticos/metabolismo , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Purinas/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Roscovitina , Serina/metabolismo , Ovinos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sinaptossomos/química , Sinaptossomos/metabolismo , Sinaptossomos/ultraestrutura
10.
Cell Mol Life Sci ; 67(21): 3725-37, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20496096

RESUMO

Successful completion of cytokinesis requires the spatio-temporal regulation of protein phosphorylation and the coordinated activity of protein kinases and phosphatases. Many mitotic protein kinases are well characterized while mitotic phosphatases are largely unknown. Here, we show that the Ca(2+)- and calmodulin-dependent phosphatase, calcineurin (CaN), is required for cytokinesis in mammalian cells, functioning specifically at the abscission stage. CaN inhibitors induce multinucleation in HeLa cells and prolong the time cells spend connected via an extended intracellular bridge. Upon Ca(2+) influx during cytokinesis, CaN is activated, targeting a set of proteins for dephosphorylation, including dynamin II (dynII). At the intracellular bridge, phospho-dynII and CaN are co-localized to dual flanking midbody rings (FMRs) that reside on either side of the central midbody ring. CaN activity and disassembly of the FMRs coincide with abscission. Thus, CaN activity at the midbody plays a key role in regulating the completion of cytokinesis in mammalian cells.


Assuntos
Calcineurina/metabolismo , Citocinese , Calcineurina/análise , Inibidores de Calcineurina , Cálcio/metabolismo , Linhagem Celular Tumoral , Dinamina II/análise , Dinamina II/metabolismo , Células HeLa , Humanos
11.
Metallomics ; 13(6)2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-33960390

RESUMO

A significant gap in the knowledge of zinc homeostasis exists for breast cancer cells. In this study, we investigated the transcriptomic response of the luminal breast cancer cells (MCF-7) to the exposure of extracellular zinc using next-generation RNA sequencing. The dataset was collected for three time points (T0, T30, and T120) in the time course of zinc treatment, which revealed the dramatic increase, up to 869-fold, of the gene expression for metallothioneins (MT1B, MT1F, MT1X, and MT2A) and the zinc exporter ZnT1 (SLC30A1) at T30, continuingly through to T120. The similar dynamic expression pattern was found for the autophagy-related gene (VMP1) and numerous genes for zinc finger proteins (e.g. RNF165, ZNF365, ZBTB2, SNAI1, ZNF442, ZNF547, ZNF563, and ZNF296). These findings point to the all-hands-on-deck strategy adopted by the cancer cells for maintaining zinc homeostasis. The stress responsive genes encoding heat shock proteins (HSPA1A, HSPA1B, HSPA1L, HSPA4L, HSPA6, HSPA8, HSPH1, HSP90AA1, and HSP90AB1) and the MTF-1 biomarker genes (AKR1C2, CLU, ATF3, GDF15, HMOX1, MAP1A, MAFG, SESN2, and UBC) were also differentially up-regulated at T120, suggesting a role of heat shock proteins and the MTF-1 related stress proteins in dealing with zinc exposure. It is for the first time that the gene encoding Polo-like kinase 2 (PLK2) was found to be involved in zinc-related response. The top differentially expressed genes were validated by qRT-PCR and further extended to the basal type breast cancer cells (MDA-MB-231). It was found that the expression level of SLC30A1 in MDA-MB-231 was higher than MCF-7 in response to zinc exposure. Taken together, the findings contribute to our knowledge and understanding of zinc homeostasis in breast cancer cells.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Homeostase , Transcriptoma/efeitos dos fármacos , Zinco/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células , Feminino , Humanos , Células MCF-7
12.
Mol Pharmacol ; 72(6): 1425-39, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17702890

RESUMO

Dynamin is a GTPase enzyme involved in membrane constriction and fission during endocytosis. Phospholipid binding via its pleckstrin homology domain maximally stimulates dynamin activity. We developed a series of surface-active small-molecule inhibitors, such as myristyl trimethyl ammonium bromide (MiTMAB) and octadecyltrimethyl ammonium bromide (OcTMAB), and we now show MiTMAB targets the dynamin-phospholipid interaction. MiTMAB inhibited dynamin GTPase activity, with a Ki of 940 +/- 25 nM. It potently inhibited receptor-mediated endocytosis (RME) of transferrin or epidermal growth factor (EGF) in a range of cells without blocking EGF binding, receptor number, or autophosphorylation. RME inhibition was rapidly reversed after washout. The rank order of potency for a variety of MiTMAB analogs on RME matched the rank order for dynamin inhibition, suggesting dynamin recruitment to the membrane is a primary cellular target. MiTMAB also inhibited synaptic vesicle endocytosis in rat brain nerve terminals (synaptosomes) without inducing depolarization or morphological defects. Therefore, the drug rapidly and reversibly blocks multiple forms of endocytosis with no acute cellular damage. The unique mechanism of action of MiTMAB provides an important tool to better understand dynamin-mediated membrane trafficking events in a variety of cells.


Assuntos
Alcanos/farmacologia , Dinamina II/antagonistas & inibidores , Dinamina I/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Compostos de Amônio Quaternário/farmacologia , Compostos de Trimetil Amônio/farmacologia , Alcanos/química , Animais , Células COS , Chlorocebus aethiops , Dinamina I/fisiologia , Dinamina II/fisiologia , Células HeLa , Humanos , Compostos de Amônio Quaternário/química , Ovinos , Tensoativos/química , Tensoativos/farmacologia , Compostos de Trimetil Amônio/química
13.
DNA Cell Biol ; 33(7): 399-407, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24673574

RESUMO

Mutations in serine palmitoyltransferase long chain subunit 1 (SPTLC1) cause the typical length-dependent axonal degeneration hereditary sensory neuropathy type 1 (HSN1). Transmission electron microscopy studies on SPTLC1 mutant lymphoblasts derived from patients revealed specific structural abnormalities of mitochondria. Swollen mitochondria with abnormal cristae were clustered around the nucleus, with some mitochondria being wrapped in rough endoplasmic reticulum (ER) membranes. Total mitochondrial counts revealed a significant change in mitochondrial numbers between healthy and diseased lymphocytes but did not reveal any change in length to width ratios nor were there any changes to cellular function. However, there was a notable change in ER homeostasis, as assessed using key ER stress markers, BiP and ERO1-Lα, displaying reduced protein expression. The observations suggest that SPTLC1 mutations cause mitochondrial abnormalities and ER stress in HSN1 cells.


Assuntos
Estresse do Retículo Endoplasmático/genética , Linfócitos/patologia , Mitocôndrias/química , Mitocôndrias/genética , Serina C-Palmitoiltransferase/genética , Western Blotting , Citometria de Fluxo , Humanos , Linfócitos/ultraestrutura , Microscopia Confocal , Mitocôndrias/ultraestrutura , Mutação/genética
14.
Nat Protoc ; 7(8): 1439-55, 2012 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-22767087

RESUMO

Small molecules modulating synaptic vesicle endocytosis (SVE) may ultimately be useful for diseases where pathological neurotransmission is implicated. Only a small number of specific SVE modulators have been identified to date. Slow progress is due to the laborious nature of traditional approaches to study SVE, in which nerve terminals are identified and studied in cultured neurons, typically yielding data from 10-20 synapses per experiment. We provide a protocol for a quantitative, high-throughput method for studying SVE in thousands of nerve terminals. Rat forebrain synaptosomes are attached to 96-well microplates and depolarized; SVE is then quantified by uptake of the dye FM4-64, which is imaged by high-content screening. Synaptosomes that have been frozen and stored can be used in place of fresh synaptosomes, reducing the experimental time and animal numbers required. With a supply of frozen synaptosomes, the assay can be performed within a day, including data analysis.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Biologia Molecular/métodos , Vesículas Sinápticas/fisiologia , Sinaptossomos/metabolismo , Animais , Corantes Fluorescentes/análise , Corantes Fluorescentes/farmacocinética , Congelamento , Ensaios de Triagem em Larga Escala/instrumentação , Masculino , Biologia Molecular/instrumentação , Prosencéfalo/citologia , Compostos de Piridínio/análise , Compostos de Piridínio/farmacocinética , Compostos de Amônio Quaternário/análise , Compostos de Amônio Quaternário/farmacocinética , Ratos , Ratos Sprague-Dawley
15.
J Biol Chem ; 278(31): 29065-71, 2003 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12754199

RESUMO

Maintaining synaptic transmission requires replenishment of docked synaptic vesicles within the readily releasable pool (RRP) from synaptic vesicle clusters in the synapsin-bound reserve pool. We show that synapsin forms a complex with phosphatidylinositol 3-kinase (PI 3-kinase) in intact nerve terminals and that synapsin-associated kinase activity increases on depolarization. Disruption of either PI 3-kinase activity or its interaction with synapsin inhibited replenishment of the RRP, but did not affect exocytosis from the RRP. Thus we conclude that a synapsin-associated PI 3-kinase activity plays a role in synaptic vesicle delivery to the RRP. This also suggests that PI 3-kinase contributes to the maintenance of synaptic transmission during periods of high activity, indicating a possible role in synaptic plasticity.


Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/fisiologia , Androstadienos/farmacologia , Animais , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Exocitose , Ácido Glutâmico/metabolismo , Morfolinas/farmacologia , Terminações Nervosas/metabolismo , Plasticidade Neuronal , Inibidores de Fosfoinositídeo-3 Quinase , Ratos , Transmissão Sináptica/fisiologia , Wortmanina
16.
J Neurochem ; 91(3): 579-90, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15485489

RESUMO

The septins are GTPase enzymes with multiple roles in cytokinesis, cell polarity or exocytosis. The proteins from the mammalian septin genes are called Sept1-10. Most are expressed in multiple tissues, but the mRNA for Sept5 (CDCrel-1) and Sept3 (G-septin) appear to be primarily expressed in brain. Sept3 is phosphorylated by cGMP-dependent protein kinase I (PKG-I) and the cGMP/PKG pathway is involved in presynaptic plasticity. Therefore to determine whether Sept3 specifically associates with neurones and nerve terminals we investigated its distribution in rat brain and neuronal cultures. Sept3 protein was detected only in brain by immunoblot, but not in 12 other tissues examined. Levels were high in all adult brain regions, and reduced in those enriched in white matter. Expression was developmentally regulated, being absent in the early embryo, low in late embryonic rat brain and increasing after birth. Like dynamin I, Sept3 was specifically enriched in synaptosomes compared with whole brain, and was only found in a peripheral membrane extract and not in the soluble or membrane extracts. Sept3 was particularly abundant in mossy fibre nerve terminals in the hippocampus. In primary cultured hippocampal neurones Sept3 immunoreactivity was punctate in neurites and predominantly localized to presynaptic terminals, strongly colocalizing with synaptophysin and dynamin I. The specific nerve terminal localization was confirmed by immunogold electron microscopy. Together this shows that Sept3 is a neurone-specific protein highly enriched in nerve terminals which supports a secretory role in synaptic vesicle recycling.


Assuntos
Encéfalo/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/metabolismo , Fosfoproteínas/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Química Encefálica , Células Cultivadas , Dinamina I/metabolismo , Hipocampo/citologia , Hipocampo/embriologia , Immunoblotting , Masculino , Microscopia Imunoeletrônica , Fibras Musgosas Hipocampais/metabolismo , Neurônios/citologia , Especificidade de Órgãos , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Septinas , Sinaptofisina/metabolismo , Sinaptossomos/química , Sinaptossomos/metabolismo
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