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1.
FASEB J ; 35(7): e21691, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34118085

RESUMO

Amyloid ß peptide (Aß) is the major pathogenic molecule in Alzheimer's disease (AD). BACE1 enzyme is essential for the generation of Aß. Deficiency of p38α-MAPK in neurons increases lysosomal degradation of BACE1 and decreases Aß deposition in the brain of APP-transgenic mice. However, the mechanisms mediating effects of p38α-MAPK are largely unknown. In this study, we used APP-transgenic mice and cultured neurons and observed that deletion of p38α-MAPK specifically in neurons decreased phosphorylation of Snapin at serine, increased retrograde transportation of BACE1 in axons and reduced BACE1 at synaptic terminals, which suggests that p38α-MAPK deficiency promotes axonal transportation of BACE1 from its predominant locations, axonal terminals, to lysosomes in the cell body. In vitro kinase assay revealed that p38α-MAPK directly phosphorylates Snapin. By further performing mass spectrometry analysis and site-directed mutagenic experiments in SH-SY5Y cell lines, we identified serine residue 112 as a p38α-MAPK-phosphorylating site on Snapin. Replacement of serine 112 with alanine did abolish p38α-MAPK knockdown-induced reduction of BACE1 activity and protein level, and transportation to lysosomes in SH-SY5Y cells. Taken together, our study suggests that activation of p38α-MAPK phosphorylates Snapin and inhibits the retrograde transportation of BACE1 in axons, which might exaggerate amyloid pathology in AD brain.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/fisiologia , Ácido Aspártico Endopeptidases/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Presenilina-1/fisiologia , Terminações Pré-Sinápticas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Transporte Axonal , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Proteína Quinase 14 Ativada por Mitógeno/genética , Neurônios/citologia , Neurônios/metabolismo , Proteínas de Transporte Vesicular/genética
2.
J Immunol ; 204(10): 2818-2828, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32269094

RESUMO

CTLs release cytotoxic proteins such as granzymes and perforin through fusion of cytotoxic granules (CG) at the target cell interface, the immune synapse, to kill virus-infected and tumorigenic target cells. A characteristic feature of these granules is their acidic pH inside the granule lumen, which is required to process precursors of granzymes and perforin to their mature form. However, the role of acidic pH in CG maturation, transport, and fusion is not understood. We demonstrate in primary murine CTLs that the a3-subunit of the vacuolar-type (H+)-adenosine triphosphatase is required for establishing a luminal pH of 6.1 inside CG using ClopHensorN(Q69M), a newly generated CG-specific pH indicator. Knockdown of the a3-subunit resulted in a significantly reduced killing of target cells and a >50% reduction in CG fusion in total internal reflection fluorescence microscopy, which was caused by a reduced number of CG at the immune synapse. Superresolution microscopy revealed a reduced interaction of CG with the microtubule network upon a3-subunit knockdown. Finally, we find by electron and structured illumination microscopy that knockdown of the a3-subunit altered the diameter and density of individual CG, whereas the number of CG per CTL was unaffected. We conclude that the a3-subunit of the vacuolar adenosine triphosphatase is not only responsible for the acidification of CG, but also contributes to the maturation and efficient transport of the CG to the immune synapse.


Assuntos
Sinapses Imunológicas/metabolismo , Microtúbulos/metabolismo , Vesículas Secretórias/metabolismo , Linfócitos T Citotóxicos/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Células Cultivadas , Citotoxicidade Imunológica , Exocitose , Concentração de Íons de Hidrogênio , Sinapses Imunológicas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Proteínas R-SNARE/genética , Linfócitos T Citotóxicos/imunologia , ATPases Vacuolares Próton-Translocadoras/genética
3.
J Neurosci ; 39(1): 18-27, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30389842

RESUMO

The calcium-dependent activator proteins for secretion (CAPS) are priming factors for synaptic and large dense-core vesicles (LDCVs), promoting their entry into and stabilizing the release-ready state. A modulatory role of CAPS in catecholamine loading of vesicles has been suggested. Although an influence of CAPS on monoamine transporter function and on vesicle acidification has been reported, a role of CAPS in vesicle loading is disputed. Using expression of naturally occurring splice variants of CAPS2 into chromaffin cells from CAPS1/CAPS2 double-deficient mice of both sexes, we show that an alternative exon of 40 aa is responsible for enhanced catecholamine loading of LDCVs in mouse chromaffin cells. The presence of this exon leads to increased activity of both vesicular monoamine transporters. Deletion of CAPS does not alter acidification of vesicles. Our results establish a splice-variant-dependent modulatory effect of CAPS on catecholamine content in LDCVs.SIGNIFICANCE STATEMENT The calcium activator protein for secretion (CAPS) promotes and stabilizes the entry of catecholamine-containing vesicles of the adrenal gland into a release-ready state. Expression of an alternatively spliced exon in CAPS leads to enhanced catecholamine content in chromaffin granules. This exon codes for 40 aa with a high proline content, consistent with an unstructured loop present in the portion of the molecule generally thought to be involved in vesicle priming. CAPS variants containing this exon promote serotonin uptake into Chinese hamster ovary cells expressing either vesicular monoamine transporter. Epigenetic tuning of CAPS variants may allow modulation of endocrine adrenaline and noradrenaline release. This mechanism may extend to monoamine release in central neurons or in the enteric nervous system.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/fisiologia , Catecolaminas/metabolismo , Células Cromafins/metabolismo , Vesículas Citoplasmáticas/metabolismo , Éxons/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Feminino , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Serotonina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo
4.
Int J Mol Sci ; 21(13)2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32629968

RESUMO

Cytotoxic T lymphocytes (CTL) are an essential part of our immune system by killing infected and malignant cells. To fully understand this process, it is necessary to study CTL function in the physiological setting of a living organism to account for their interplay with other immune cells like CD4+ T helper cells and macrophages. The anterior chamber of the eye (ACE), originally developed for diabetes research, is ideally suited for non-invasive and longitudinal in vivo imaging. We take advantage of the ACE window to observe immune responses, particularly allorejection of islets of Langerhans cells by CTLs. We follow the onset of the rejection after vascularization on islets until the end of the rejection process for about a month by repetitive two-photon microscopy. We find that CTLs show reduced migration on allogeneic islets in vivo compared to in vitro data, indicating CTL activation. Interestingly, the temporal infiltration pattern of T cells during rejection is precisely regulated, showing enrichment of CD4+ T helper cells on the islets before arrival of CD8+ CTLs. The adaptation of the ACE to immune responses enables the examination of the mechanism and regulation of CTL-mediated killing in vivo and to further investigate the killing in gene-deficient mice that resemble severe human immune diseases.


Assuntos
Câmara Anterior/imunologia , Rejeição de Enxerto/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Linfócitos T Citotóxicos/fisiologia , Animais , Camundongos Endogâmicos DBA
5.
Int J Mol Sci ; 21(7)2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-32252488

RESUMO

Cytotoxic T lymphocytes (CTL) are key players of the adaptive immune system that target tumors and infected cells. A central step to that is the formation of a cell-cell contact zone between the CTL and its target called an immune synapse (IS). Here, we investigate the influence of the initial T cell receptor (TCR) trigger of a cytolytic IS on the distinct steps leading to cytotoxic granule (CG) exocytosis. We stimulated primary CTLs from mouse using lipid bilayers with varying anti-CD3 but constant ICAM concentrations. We fluorescently labeled molecular markers of distinct IS zones such as actin, CD3, granzyme B, and Synaptobrevin2 in CTLs and imaged cytolytic IS formation by total internal reflection fluorescence microscopy (TIRFM). We found that an intermediate anti-CD3 concentration of 10 µg/mL induces the fastest adhesion of CTLs to the bilayers and results in maximal CG fusion efficiency. The latency of actin ring formation, dwell time, and maximum surface area at the IS exhibit different dependencies on the stimulatory anti-CD3 concentrations. The number and surface area of CD3 clusters at the IS seem to show a different dependency to the TCR trigger when compared to their dwell time. Finally, the mode of full CG exocytosis appears to be independent of the TCR trigger.


Assuntos
Sinapses Imunológicas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Biomarcadores , Degranulação Celular/imunologia , Citotoxicidade Imunológica , Exocitose/imunologia , Ativação Linfocitária/imunologia , Camundongos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo
6.
J Immunol ; 197(6): 2473-84, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27527597

RESUMO

CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process.


Assuntos
Grânulos Citoplasmáticos/imunologia , Endocitose , Linfócitos T Citotóxicos/imunologia , Animais , Clatrina/metabolismo , Grânulos Citoplasmáticos/fisiologia , Dinaminas/imunologia , Dinaminas/metabolismo , Endossomos/imunologia , Endossomos/metabolismo , Exocitose , Granzimas/metabolismo , Sinapses Imunológicas , Camundongos , Proteínas R-SNARE/imunologia
7.
Cell Mol Life Sci ; 74(3): 399-408, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27585956

RESUMO

Cytotoxic T lymphocytes patrol our body in search for infected cells which they kill through the release of cytotoxic substances contained in cytotoxic granules. The fusion of cytotoxic granules occurs at a specially formed contact site, the immunological synapse, and is tightly controlled to ensure specificity. In this review, we discuss the contribution of two intracellular compartments, endosomes and cytotoxic granules, to the formation, function and disassembly of the immunological synapse. We highlight a recently proposed sequential process of fusion events at the IS upon target cell recognition. First, recycling endosomes fuse with the plasma membrane to deliver cargo required for the docking of cytotoxic granules. Second, cytotoxic granules arrive and fuse upon docking in a SNARE-dependent manner. Following fusion, membrane components of the cytotoxic granule are retrieved through endocytosis to ensure the fast, efficient serial killing of target cells that is characteristic of cytotoxic T lymphocytes.


Assuntos
Citotoxicidade Imunológica , Endocitose , Exocitose , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Animais , Grânulos Citoplasmáticos/imunologia , Endossomos/imunologia , Humanos , Lisossomos/imunologia , Fusão de Membrana , Proteínas SNARE/imunologia
8.
J Neurosci ; 36(8): 2473-93, 2016 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-26911694

RESUMO

Mutations in the Tulp1 gene cause severe, early-onset retinitis pigmentosa (RP14) in humans. In the retina, Tulp1 is mainly expressed in photoreceptors that use ribbon synapses to communicate with the inner retina. In the present study, we demonstrate that Tulp1 is highly enriched in the periactive zone of photoreceptor presynaptic terminals where Tulp1 colocalizes with major endocytic proteins close to the synaptic ribbon. Analyses of Tulp1 knock-out mice demonstrate that Tulp1 is essential to keep endocytic proteins enriched at the periactive zone and to maintain high levels of endocytic activity close to the synaptic ribbon. Moreover, we have discovered a novel interaction between Tulp1 and the synaptic ribbon protein RIBEYE, which is important to maintain synaptic ribbon integrity. The current findings suggest a new model for Tulp1-mediated localization of the endocytic machinery at the periactive zone of ribbon synapses and offer a new rationale and mechanism for vision loss associated with genetic defects in Tulp1.


Assuntos
Endocitose/fisiologia , Proteínas do Olho/metabolismo , Células Fotorreceptoras/metabolismo , Sinapses/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Proteínas do Olho/análise , Proteínas do Olho/genética , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Técnicas de Cultura de Órgãos , Células Fotorreceptoras/química , Retina/química , Retina/metabolismo , Sinapses/química , Sinapses/genética
9.
Biochim Biophys Acta ; 1863(7 Pt A): 1653-64, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27094127

RESUMO

Cytotoxic T lymphocytes (CTL) eliminate pathogen-infected and cancerous cells mainly by polarized secretion of lytic granules (LG, containing cytotoxic molecules like perforin and granzymes) at the immunological synapse (IS). Members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family are involved in trafficking (generation, transport and fusion) of vesicles at the IS. Syntaxin 8 (Stx8) is expressed in LG and colocalizes with the T cell receptor (TCR) upon IS formation. Here, we report the significance of Stx8 for human CTL cytotoxicity. We found that Stx8 mostly localized in late, recycling endosomal and lysosomal compartments with little expression in early endosomal compartments. Down-regulation of Stx8 by siRNA resulted in reduced cytotoxicity. We found that following perforin release of the pre-existing pool upon target cell contact, Stx8 down-regulated CTL regenerate perforin pools less efficiently and thus release less perforin compared to control CTL. CD107a degranulation, real-time and end-point population cytotoxicity assays, and high resolution microscopy support our conclusion that Stx8 is required for proper and timely sorting and trafficking of cytotoxic molecules to functional LG through the endosomal pathway in human CTL.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas Qa-SNARE/metabolismo , Vesículas Secretórias/metabolismo , Linfócitos T Citotóxicos/metabolismo , Degranulação Celular , Linhagem Celular , Grânulos Citoplasmáticos/imunologia , Citotoxicidade Imunológica , Endossomos/metabolismo , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Perforina/metabolismo , Transporte Proteico , Proteínas Qa-SNARE/genética , Interferência de RNA , Vesículas Secretórias/imunologia , Linfócitos T Citotóxicos/imunologia , Fatores de Tempo , Transfecção
10.
J Neurochem ; 137(6): 849-59, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26938142

RESUMO

Exocytosis is the process by which stored neurotransmitters and hormones are released via the fusion of secretory vesicles with the plasma membrane. It is a dynamic, rapid and spatially restricted process involving multiple steps including vesicle trafficking, tethering, docking, priming and fusion. For many years great steps have been undertaken in our understanding of how exocytosis occurs in different cell types, with significant focus being placed on synaptic release and neurotransmission. However, this process of exocytosis is an essential component of cell signalling throughout the body and underpins a diverse array of essential physiological pathways. Many similarities exist between different cell types with regard to key aspects of the exocytosis pathway, such as the need for Ca(2+) to trigger it or the involvement of members of the N-ethyl maleimide-sensitive fusion protein attachment protein receptor protein families. However, it is also equally clear that non-neuronal cells have acquired highly specialized mechanisms to control the release of their own unique chemical messengers. This review will focus on several important non-neuronal cell types and discuss what we know about the mechanisms they use to control exocytosis and how their specialized output is relevant to the physiological role of each individual cell type. These include enteroendocrine cells, pancreatic ß cells, astrocytes, lactotrophs and cytotoxic T lymphocytes. Non-neuronal cells have acquired highly specialized mechanisms to control the release of unique chemical messengers, such as polarised fusion of insulin granules in pancreatic ß cells targeted towards the vasculature (top). This review discusses mechanisms used in several important non-neuronal cell types to control exocytosis, and the relevance of intermediate vesicle fusion pore states (bottom) and their specialized output to the physiological role of each cell type. These include enteroendocrine cells, pancreatic ß cells, astrocytes, lactotrophs and cytotoxic T lymphocytes. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).


Assuntos
Sistema Endócrino/citologia , Exocitose/fisiologia , Neuroglia/fisiologia , Vesículas Secretórias/fisiologia , Animais , Membrana Celular , Fusão de Membrana/fisiologia , Proteínas do Tecido Nervoso/metabolismo
11.
J Immunol ; 193(6): 3146-54, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25122923

RESUMO

In a previous study, we identified thioredoxin domain containing 16 (TXNDC16) as a meningioma-associated Ag by protein macroarray screening. Serological screening detected autoantibodies against TXNDC16 exclusively in meningioma patients' sera and not in sera of healthy controls. TXNDC16 was previously found to be an endoplasmic reticulum (ER)-luminal glycoprotein. In this study, we show an additional ER-associated localization of TXNDC16 in the cytosol by in vitro synthesis, molecular mass shift assay, and flow cytometry. We were able to show TXNDC16 secretion in different human cell lines due to masked and therefore nonfunctional ER retrieval motif. A previously indicated exosomal TXNDC16 secretion could not be confirmed in HEK293 cells. The secreted serum protein TXNDC16 is bound in circulating immune complexes, which were found both in meningioma and healthy blood donor sera. Employing a customized array with 163 overlapping TXNDC16 peptides and measuring autoantibody reactivity, we achieved discrimination of meningioma sera from healthy controls with an accuracy of 87.2% using a set of only five immunogenic TXNDC16 epitopes.


Assuntos
Complexo Antígeno-Anticorpo/sangue , Antígenos de Neoplasias/imunologia , Glicoproteínas de Membrana/imunologia , Meningioma/imunologia , Sequência de Aminoácidos , Autoanticorpos/sangue , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Epitopos/imunologia , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular
12.
Traffic ; 14(7): 798-809, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23590328

RESUMO

In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion-competent through the priming process. In secretory tissues such as brain and neuroendocrine glands, this process is mediated by members of the Munc13 protein family. In human CTLs, mutations in the Munc13-4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3, suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs at both the mRNA and protein level. We demonstrate that Munc13-1 and Munc13-4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13-4-deficient Jinx mice. Mobility studies using total internal reflection fluorescence microscopy indicate that Munc13-4 and Munc13-1 are responsible for the priming process of LGs. Furthermore, the domains of the Munc13 protein, which is responsible for functional fusion, could be identified. We conclude from these data that both isoforms of the Munc13 family, Munc13-1 and Munc13-4, are functionally redundant in murine CTLs.


Assuntos
Exocitose , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Vesículas Secretórias/metabolismo , Linfócitos T Citotóxicos/metabolismo , Animais , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas do Tecido Nervoso/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína
13.
EMBO J ; 30(19): 3895-912, 2011 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-21847095

RESUMO

Cell polarization enables restriction of signalling into microdomains. Polarization of lymphocytes following formation of a mature immunological synapse (IS) is essential for calcium-dependent T-cell activation. Here, we analyse calcium microdomains at the IS with total internal reflection fluorescence microscopy. We find that the subplasmalemmal calcium signal following IS formation is sufficiently low to prevent calcium-dependent inactivation of ORAI channels. This is achieved by localizing mitochondria close to ORAI channels. Furthermore, we find that plasma membrane calcium ATPases (PMCAs) are re-distributed into areas beneath mitochondria, which prevented PMCA up-modulation and decreased calcium export locally. This nano-scale distribution-only induced following IS formation-maximizes the efficiency of calcium influx through ORAI channels while it decreases calcium clearance by PMCA, resulting in a more sustained NFAT activity and subsequent activation of T cells.


Assuntos
Sinalização do Cálcio , Cálcio/química , Linfócitos T/citologia , Canais de Cálcio/metabolismo , Membrana Celular/enzimologia , Citoesqueleto/metabolismo , Eletrofisiologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Sinapses Imunológicas , Células Jurkat , Ativação Linfocitária , Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , Proteína ORAI1 , Estrutura Terciária de Proteína
14.
Eur J Immunol ; 44(2): 573-84, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24227526

RESUMO

CTLs kill target cells via fusion of lytic granules (LGs) at the immunological synapse (IS). Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) function as executors of exocytosis. The importance of SNAREs in CTL function is evident in the form of familial hemophagocytic lymphohistiocytosis type 4 that is caused by mutations in Syntaxin11 (Stx11), a Qa-SNARE protein. Here, we investigate the molecular mechanism of Stx11 function in primary human effector CTLs with high temporal and spatial resolution. Downregulation of endogenous Stx11 resulted in a complete inhibition of LG fusion that was paralleled by a reduction in LG dwell time at the IS. Dual color evanescent wave imaging suggested a sequential process, in which first Stx11 is transported to the IS through a subpopulation of recycling endosomes. The resulting Stx11 clusters at the IS then serve as a platform to mediate fusion of arriving LGs. We conclude that Stx11 functions as a t-SNARE for the final fusion of LG at the IS, explaining the severe phenotype of familial hemophagocytic lymphohistiocytosis type 4 on a molecular level.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/metabolismo , Linfócitos T Citotóxicos/metabolismo , Células Cultivadas , Grânulos Citoplasmáticos/imunologia , Regulação para Baixo/imunologia , Endossomos/imunologia , Endossomos/metabolismo , Humanos , Sinapses Imunológicas/imunologia , Sinapses Imunológicas/metabolismo , Linfo-Histiocitose Hemofagocítica/imunologia , Linfo-Histiocitose Hemofagocítica/metabolismo , Proteínas Qa-SNARE/imunologia , Proteínas SNARE/imunologia , Linfócitos T Citotóxicos/imunologia
16.
J Neurosci ; 33(43): 17123-37, 2013 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-24155316

RESUMO

Large dense core vesicle (LDCV) exocytosis in chromaffin cells follows a well characterized process consisting of docking, priming, and fusion. Total internal reflection fluorescence microscopy (TIRFM) studies suggest that some LDCVs, although being able to dock, are resistant to calcium-triggered release. This phenomenon termed dead-end docking has not been investigated until now. We characterized dead-end vesicles using a combination of membrane capacitance measurement and visualization of LDCVs with TIRFM. Stimulation of bovine chromaffin cells for 5 min with 6 µm free intracellular Ca2+ induced strong secretion and a large reduction of the LDCV density at the plasma membrane. Approximately 15% of the LDCVs were visible at the plasma membrane throughout experiments, indicating they were permanently docked dead-end vesicles. Overexpression of Munc18-2 or SNAP-25 reduced the fraction of dead-end vesicles. Conversely, expressing open-syntaxin increased the fraction of dead-end vesicles. These results indicate the existence of the unproductive target soluble N-ethylmaleimide-sensitive factor attachment protein receptor acceptor complex composed of 2:1 syntaxin-SNAP-25 in vivo. More importantly, they define a novel function for this acceptor complex in mediating dead-end docking.


Assuntos
Membrana Celular/metabolismo , Células Cromafins/metabolismo , Vesículas Secretórias/metabolismo , Animais , Cálcio/metabolismo , Bovinos , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Proteínas Q-SNARE/genética , Proteínas Q-SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo
17.
J Cell Sci ; 125(Pt 8): 1958-69, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22375059

RESUMO

Co-translational transport of polypeptides into the endoplasmic reticulum (ER) involves the Sec61 channel and additional components such as the ER lumenal Hsp70 BiP and its membrane-resident co-chaperone Sec63p in yeast. We investigated whether silencing the SEC61A1 gene in human cells affects co- and post-translational transport of presecretory proteins into the ER and post-translational membrane integration of tail-anchored proteins. Although silencing the SEC61A1 gene in HeLa cells inhibited co- and post-translational transport of signal-peptide-containing precursor proteins into the ER of semi-permeabilized cells, silencing the SEC61A1 gene did not affect transport of various types of tail-anchored protein. Furthermore, we demonstrated, with a similar knockdown approach, a precursor-specific involvement of mammalian Sec63 in the initial phase of co-translational protein transport into the ER. By contrast, silencing the SEC62 gene inhibited only post-translational transport of a signal-peptide-containing precursor protein.


Assuntos
DNA Helicases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos/metabolismo , Animais , DNA Helicases/genética , Retículo Endoplasmático/genética , Inativação Gênica , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Camundongos , Chaperonas Moleculares , Células NIH 3T3 , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas de Ligação a RNA , Canais de Translocação SEC
18.
Traffic ; 12(7): 890-901, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21438968

RESUMO

SNARE proteins are essential fusion mediators for many intracellular trafficking events. Here, we investigate the role of Syntaxin7 (Stx7) in the release of lytic granules from cytotoxic T lymphocytes (CTLs). We show that Stx7 is expressed in CTLs and is preferentially localized to the region of lytic granule release, the immunological synapse (IS). Interference of Stx7 function by expression of a dominant-negative Stx7 construct or by small interfering RNA leads to a dramatic reduction of CTL-mediated killing of target cells. Real-time visualization of individual lytic granules at the IS by evanescent wave microscopy reveals that lytic granules in Stx7-deprived CTLs not only fail to fuse with the plasma membrane but even fail to accumulate at the IS. Surprisingly, the accumulation defect is not caused by an overall reduction in lytic granule number, but by a defect in the trafficking of T cell receptors (TCRs) through endosomes. Subsequent high-resolution nanoscopy shows that Stx7 colocalizes with Rab7 on late endosomes. We conclude from these data that the accumulation of recycling TCRs at the IS is a SNARE-dependent process and that Stx7-mediated processing of recycling TCRs through endosomes is a prerequisite for the cytolytic function of CTLs.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Exocitose/fisiologia , Proteínas Qa-SNARE/metabolismo , Linfócitos T Citotóxicos/metabolismo , Biomarcadores/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Endossomos/metabolismo , Exocitose/imunologia , Humanos , Sinapses Imunológicas/fisiologia , Ativação Linfocitária , Fusão de Membrana/fisiologia , Proteínas Qa-SNARE/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/citologia
19.
Eur J Immunol ; 42(2): 470-5, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22120889

RESUMO

The major function of cytotoxic T lymphocytes (CTLs) is to eliminate pathogen-infected and tumorigenic cells. This is mediated mainly through the exocytosis of lytic granules (LGs) containing cytotoxic components, such as perforin and granzymes at the immunological synapse (IS). The soluble NSF attachment receptor (SNARE) protein isoforms are well known to be required for vesicle exocytosis in neuronal synapses, but their potential function in CTLs is only partly understood. Here, we examined the expression of SNARE proteins before and after the activation of primary human CD8(+) T cells and determined their co-localization with LGs and CD3 after IS formation with target cells. We found that several key SNARE proteins in neuronal cells were not expressed in CTLs, such as syntaxin1B2 and SNAP-25. Vti1b, Stx8 and Stx16 had the highest degrees of co-localization with LGs while Stx3, Stx4, Stx6, Stx7, Stx8, Stx13, Vti1b, VAMP3 and VAMP4 co-localized with CD3. Our data provide the first complete expression profile and localization of SNAREs in primary human CD8(+) T cells, laying the groundwork for further understanding their potential role in T-cell function.


Assuntos
Complexo CD3/metabolismo , Sinapses Imunológicas/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas SNARE/metabolismo , Linfócitos T Citotóxicos/metabolismo , Células Cultivadas , Citotoxicidade Imunológica , Exocitose/imunologia , Humanos , Ativação Linfocitária , Neurônios/imunologia , Neurônios/metabolismo , Neurônios/patologia , Especificidade de Órgãos , Perforina/metabolismo , Transporte Proteico , Vesículas Secretórias/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia
20.
J Immunol ; 186(12): 6894-904, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21562157

RESUMO

Lytic granule (LG)-mediated apoptosis is the main mechanism by which CTL kill virus-infected and tumorigenic target cells. CTL form a tight junction with the target cells, which is called the immunological synapse (IS). To avoid unwanted killing of neighboring cells, exocytosis of lytic granules (LG) is tightly controlled and restricted to the IS. In this study, we show that in activated human primary CD8(+) T cells, docking of LG at the IS requires tethering LG with CD3-containing endosomes (CD3-endo). Combining total internal reflection fluorescence microscopy and fast deconvolution microscopy (both in living cells) with confocal microscopy (in fixed cells), we found that LG and CD3-endo tether and are cotransported to the IS. Paired but not single LG are accumulated at the IS. The dwell time of LG at the IS is substantially enhanced by tethering with CD3-endo, resulting in a preferential release of paired LG over single LG. The SNARE protein Vti1b is required for tethering of LG and CD3-endo. Downregulation of Vti1b reduces tethering of LG with CD3-endo. This leads to an impaired accumulation and docking of LG at the IS and a reduction of target cell killing. Therefore, Vti1b-dependent tethering of LG and CD3-endo determines accumulation, docking, and efficient lytic granule secretion at the IS.


Assuntos
Complexo CD3 , Endossomos/imunologia , Granzimas/imunologia , Sinapses Imunológicas/imunologia , Proteínas Qb-SNARE/imunologia , Linfócitos T Citotóxicos/imunologia , Células Cultivadas , Humanos , Microscopia , Ligação Proteica , Proteínas Qb-SNARE/metabolismo , Vesículas Secretórias/imunologia
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