RESUMO
Blood platelets are tiny cell fragments derived from megakaryocytes. Their primary function is to control blood vessel integrity and ensure hemostasis if a vessel wall is damaged. Circulating quiescent platelets have a flat, discoid shape maintained by a circumferential microtubule bundle, called the marginal band (MB). In the case of injury platelets are activated and rapidly adopt a spherical shape due to microtubule motor-induced elongation and subsequent coiling of the MB. Platelet activation and shape change can be transient or become irreversible. This depends on the strength of the activation stimulus, which is translated into a cytoskeletal crosstalk between microtubules, their motors and the actomyosin cortex, ensuring stimulus-response coupling. Following microtubule motor-driven disc-to-sphere transition, a strong stimulus will lead to compression of the sphere through actomyosin cortex contraction. This will concentrate the granules in the center of the platelet and accelerate their exocytosis. Once granules are released, platelets have crossed the point of no return to irreversible activation. This review summarizes the current knowledge of the molecular mechanism leading to platelet shape change, with a special emphasis on microtubules, and refers to previously published observations, which have been essential for generating an integrated view of cytoskeletal rearrangements during platelet activation.
Assuntos
Citoesqueleto de Actina/metabolismo , Plaquetas/metabolismo , Forma Celular , Microtúbulos/metabolismo , Ativação Plaquetária , Animais , Vesículas Citoplasmáticas/metabolismo , Exocitose , Humanos , Proteínas Motores Moleculares/metabolismo , Vesículas Secretórias/metabolismo , Transdução de SinaisRESUMO
Histone deacetylase 6 (HDAC6) is a unique enzyme with specific structural and functional features. It is actively or stably maintained in the cytoplasm and is the only member, within the histone deacetylase family, that harbors a full duplication of its deacetylase homology region followed by a specific ubiquitin-binding domain at the C-terminus end. Accordingly, this deacetylase functions at the heart of a cellular regulatory mechanism capable of coordinating various cellular functions largely relying on the microtubule network. Moreover, HDAC6 action as a regulator of the HSP90 chaperone activity adds to the multifunctionality of the protein, and allows us to propose a critical role for HDAC6 in mediating and coordinating various cellular events in response to different stressful stimuli.
Assuntos
Citoesqueleto/enzimologia , Histona Desacetilases/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Acetilação , Animais , Núcleo Celular/enzimologia , Inibidores Enzimáticos/uso terapêutico , Inibidores de Histona Desacetilases , Histona Desacetilases/química , Humanos , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
The tSNARE SNAP-25 is expressed in pancreatic (beta)-cells and is involved in the regulated release of insulin. It has been shown previously that SNAP-25 associates with the plasma membrane consequent to palmitoylation of one or more cysteines in the central region of the molecule. The importance of palmitolyation in the biological function of SNAP-25 in exocytosis was not addressed. Furthermore, studies on both SNAP-25 and its non-palmitoylated homologues SNAP-29 and sec9, have suggested an alternative or complementary mechanism for membrane association involving interaction with syntaxin. To address these issues, we have now studied the behavior and biological activity of cysteine mutant SNAP-25 in insulin-secreting (HIT) cells. While 91% of native SNAP-25 was associated with the membrane, this value decreased to 56% for the single cysteine mutant C85/A and to 10% for the double (C85,88/A) and quadruple (C85,88,90,92/A) mutants. The mutant SNAP-25 forms were all found to bind syntaxin 1A with equal efficacy. Over-expression of syntaxin 1A in HIT cells allowed for partial relocalization of both the double and quadruple SNAP-25 cys mutants to the membrane. By introducing a further mutation to the SNAP-25 molecules to render them resistant to botulinum neurotoxin E, it was possible to study their ability to reconstitute regulated insulin secretion in toxin-treated HIT cells. Native SNAP-25 was able to fully reconstitute secretory activity in such cells. Despite the fact that the single cysteine mutant was significantly displaced to the cytosol, it still displayed 82% activity in the secretion reconstitution assay, and a similar discrepancy was seen for the double mutant. Even the quadruple mutant with no remaining cysteines was able to support a minimal level of secretion. It is concluded that both palmitoylation and binding to syntaxin are implicated in membrane association of SNAP-25. This as well as the discrepancy between membrane localization and biological activity of the cysteine mutants, suggests a complex, multi-component process for association of SNAP-25 with the membrane and its recruitment to a biologically productive state.
Assuntos
Cisteína/metabolismo , Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/farmacologia , Cálcio/metabolismo , Linhagem Celular , Cricetinae , Cisteína/genética , Resistência a Medicamentos , Exocitose , Expressão Gênica , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/genética , Testes de Precipitina , Proteínas Qa-SNARE , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína 25 Associada a Sinaptossoma , Sintaxina 1RESUMO
The tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is N-ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in pancreatic B-cells and its cleavage by botulinum neurotoxin E (BoNT/E) abolishes stimulated secretion of insulin. In the nervous system, two SNAP-25 isoforms (a and b) have been described that are produced by alternative splicing. Here it is shown, using reverse transcriptase PCR, that messages for both SNAP-25 isoforms are expressed in primary pancreatic B and non-B cells as well as in insulin-secreting cell lines. After transfection, both isoforms can be detected at the plasma membrane as well as in an intracellular perinuclear region in the insulin-secreting cell line, HIT. To test for the functional role of the two isoforms in insulin secretion, mutant forms of SNAP-25a and b resistant against cleavage by BoNT/E were generated. Such mutant SNAP-25, when expressed in HIT cells, is not inactivated by BoNT/E and its ability to restore insulin secretion can thus be investigated. To obtain the toxin-resistant mutant isoforms, the sequence around the BoNT/E cleavage site (R176QIDRIM182) was changed to P176QIKRIT182. This is the sequence of the equivalent region of human SNAP-23 (P187-T194), which has been shown to be resistant to BoNT/E. The mutant SNAP-25 was resistant to BoNT/E in vitro and in vivo and both mutant isoforms were able to reconstitute insulin secretion from toxin-treated HIT cells.
Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Sequência de Bases , Toxinas Botulínicas/farmacologia , Linhagem Celular , Primers do DNA , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/genética , Isoformas de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteína 25 Associada a SinaptossomaRESUMO
SNAP-25 is expressed in neurons and endocrine cells and is essential for exocytosis of neurotransmitters and peptide hormones. It has been shown to be involved in several interactions with other proteins of the secretion machinery. Here we show that SNAP-25 can self-associate to form a disulfide-linked complex. Complex formation is facilitated in vitro (in concentrated extracts or by immunoprecipitation). SNAP-25 complexes, however, also form when intact cells are treated with a membrane-permeable crosslinker indicating that SNAP-25 molecules exist in close proximity in vivo and could form complexes spontaneously. We also show that monomeric SNAP-25 and disulfide-linked SNAP-25 complexes are palmitoylated and that both can be cleaved by botulinum neurotoxin E.
Assuntos
Dissulfetos/química , Proteínas de Membrana , Proteínas do Tecido Nervoso/química , Animais , Toxinas Botulínicas/química , Química Encefálica , Permeabilidade da Membrana Celular , Reagentes de Ligações Cruzadas/química , Técnicas In Vitro , Neurônios/química , Testes de Precipitina , Ratos , Proteína 25 Associada a SinaptossomaRESUMO
Dispersed rat islet cells embedded in a matrix of collagen I are known to form aggregates in vitro reminiscent of native islets. Furthermore, it appears that islet function and survival are better maintained in vitro when cells are grown in the presence of extracellular matrix. These studies suggest an important role of cell--matrix interactions in the formation and maintenance of islet structure and function. The molecular basis of these interactions is mostly unknown. In the present study, we confirm the presence of beta 1 integrins on primary and transformed (RIN-2A line) rat islet cells. Perturbation studies in vitro show that beta 1 integrins play a role in islet cell attachment and spreading on bovine extracellular matrix and on the matrix produced by A-431 cells. The alpha 3 integrin subunit is coimmunoprecipitated with beta 1 from extracts of both primary and transformed islet cells, and immunodepletion studies suggest that alpha 3 beta 1 represents nearly half of the total beta 1 integrins expressed on primary islet cells. In situ, alpha 3 and beta 1 are expressed on the surface of all islet cell types, as shown by indirect immunocytochemistry on paraformaldehyde-fixed sections of rat pancreas. In conclusion, the study demonstrates the presence of alpha 3 beta 1 on primary and transformed rat islet cells, and an important role of beta 1 integrins in islet cell attachment and spreading in vitro.
Assuntos
Antígenos CD/química , Moléculas de Adesão Celular/química , Transformação Celular Neoplásica/patologia , Integrina beta1/química , Integrinas/química , Ilhotas Pancreáticas/citologia , Animais , Antígenos CD/metabolismo , Western Blotting , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Matriz Extracelular/fisiologia , Integrina alfa3 , Integrina beta1/metabolismo , Integrinas/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/ultraestrutura , Masculino , Peso Molecular , Testes de Precipitina , Ratos , Ratos Sprague-Dawley , CalininaRESUMO
We have investigated the effect of botulinum neurotoxin (BoNT) C1 light chain (LC) on insulin exocytosis from the clonal beta-cell line HIT-T15. In streptolysin-O permeabilized cells, the beta-cell impermeant BoNT C1 cleaved mainly syntaxin 1 and inhibited Ca2+ as well as GTPgammaS induced exocytosis. To study the effect of BoNTs in intact cells, we transiently coexpressed the BoNT LC together with a reporter gene for insulin release. BoNT C1 inhibited K+ induced insulin secretion by 95% but reduced insulin release stimulated by glucose only by 25%. Thus a component of glucose stimulated insulin release is insensitive to BoNT C1.
Assuntos
Toxinas Botulínicas/farmacologia , Cálcio/farmacologia , Exocitose/efeitos dos fármacos , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Antígenos de Superfície/metabolismo , Proteínas de Bactérias , Toxinas Botulínicas/genética , Linhagem Celular , Permeabilidade da Membrana Celular , Cricetinae , Expressão Gênica , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Humanos , Secreção de Insulina , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Cloreto de Potássio/farmacologia , Proteínas R-SNARE , Proteínas Recombinantes de Fusão , Estreptolisinas/farmacologia , Proteína 25 Associada a Sinaptossoma , Sintaxina 1 , TransfecçãoRESUMO
The synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in neurons and endocrine cells. It has been shown to play an important role in the release mechanism of neurotransmitters and peptide hormones, including insulin. Thus, when insulin-secreting cells are permeabilized and treated with botulinum neurotoxin E (BoNT/E), SNAP-25 is hydrolyzed, and insulin secretion is inhibited. Recently SNAP-23, a more generally expressed isoform of SNAP-25, has been described. The functional role of SNAP-23 has not been investigated to date. It is now shown that SNAP-23 is resistant to cleavage by BoNT/E. It was therefore possible to test whether transfection of HIT (transformed pancreatic B-) cells with SNAP-23 reconstitutes insulin release from BoNT/E treated cells, in which SNAP-25 is inactivated by the toxin. The results show that SNAP-23 is able to replace SNAP-25 when it is overexpressed. While these results demonstrate that SNAP-23 is a functional homologue of SNAP-25, able to function in regulated exocytosis, they indicate that SNAP-23 may be inefficient in this process. This suggests that both isoforms may have their own specific binding partners and discrete, albeit mechanistically similar, functional roles within the cell.
Assuntos
Toxinas Botulínicas/metabolismo , Proteínas de Transporte/metabolismo , Insulina/metabolismo , Proteínas de Membrana , Proteínas do Tecido Nervoso/metabolismo , Animais , Proteínas de Transporte/genética , Cricetinae , Técnica Indireta de Fluorescência para Anticorpo , Secreção de Insulina , Proteínas do Tecido Nervoso/genética , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteína 25 Associada a Sinaptossoma , Transfecção , Células Tumorais CultivadasRESUMO
Vesicle-associated membrane protein-2 (VAMP-2) and cellubrevin are associated with the membrane of insulin-containing secretory granules and of gamma-aminobutyric acid (GABA)-containing synaptic-like vesicles of pancreatic beta-cells. We found that a point mutation in VAMP-2 preventing targeting to synaptic vesicles also impairs the localization on insulin-containing secretory granules, suggesting a similar requirement for vesicular targeting. Tetanus toxin (TeTx) treatment of permeabilized HIT-T15 cells leads to the proteolytic cleavage of VAMP-2 and cellubrevin and causes the inhibition of Ca2+-triggered insulin exocytosis. Transient transfection of HIT-T15 cells with VAMP-1, VAMP-2 or cellubrevin made resistant to the proteolytic action of TeTx by amino acid replacements in the cleavage site restored Ca2+-stimulated secretion. Wild-type VAMP-2, wild-type cellubrevin or a mutant of VAMP-2 resistant to TeTx but not targeted to secretory granules were unable to rescue Ca2+-evoked insulin release. The transmembrane domain and the N-terminal region of VAMP-2 were not essential for the recovery of stimulated exocytosis, but deletions preventing the binding to SNAP-25 and/or to syntaxin I rendered the protein inactive in the reconstitution assay. Mutations of putative phosphorylation sites or of negatively charged amino acids in the SNARE motif recognized by clostridial toxins had no effect on the ability of VAMP-2 to mediate Ca2+-triggered secretion. We conclude that: (i) both VAMP-2 and cellubrevin can participate in the exocytosis of insulin; (ii) the interaction of VAMP-2 with syntaxin and SNAP-25 is required for docking and/or fusion of secretory granules with the plasma membrane; and (iii) the phosphorylation of VAMP-2 is not essential for Ca2+-stimulated insulin exocytosis.
Assuntos
Cálcio/metabolismo , Exocitose , Insulina/metabolismo , Proteínas de Membrana/metabolismo , Toxinas Botulínicas/metabolismo , Linhagem Celular , Grânulos Citoplasmáticos/metabolismo , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Ligação Proteica , Proteínas R-SNARERESUMO
VAMP proteins are important components of the machinery controlling docking and/or fusion of secretory vesicles with their target membrane. We investigated the expression of VAMP proteins in pancreatic beta-cells and their implication in the exocytosis of insulin. cDNA cloning revealed that VAMP-2 and cellubrevin, but not VAMP-1, are expressed in rat pancreatic islets and that their sequence is identical to that isolated from rat brain. Pancreatic beta-cells contain secretory granules that store and secrete insulin as well as synaptic-like microvesicles carrying gamma-aminobutyric acid. After subcellular fractionation on continuous sucrose gradients, VAMP-2 and cellubrevin were found to be associated with both types of secretory vesicle. The association of VAMP-2 with insulin-containing granules was confirmed by confocal microscopy of primary cultures of rat pancreatic beta-cells. Pretreatment of streptolysin-O permeabilized insulin-secreting cells with tetanus and botulinum B neurotoxins selectively cleaved VAMP-2 and cellubrevin and abolished Ca(2+)-induced insulin release (IC50 approximately 15 nM). By contrast, the pretreatment with tetanus and botulinum B neurotoxins did not prevent GTP gamma S-stimulated insulin secretion. Taken together, our results show that pancreatic beta-cells express VAMP-2 and cellubrevin and that one or both of these proteins selectively control Ca(2+)-mediated insulin secretion.
Assuntos
Cálcio/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Animais , Toxinas Botulínicas/farmacologia , Química Encefálica , Fracionamento Celular , Linhagem Celular , Clonagem Molecular , Grânulos Citoplasmáticos/metabolismo , Exocitose/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Insulina/análise , Secreção de Insulina , Ilhotas Pancreáticas/química , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Proteínas R-SNARE , Ratos , Análise de Sequência de DNA , Toxina Tetânica/farmacologia , Proteína 3 Associada à Membrana da VesículaRESUMO
SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP-25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons. Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of approximately 1 and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca(++)-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of approximately 60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and half-maximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell type- the pancreatic B cell.
Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana , Proteínas do Tecido Nervoso/biossíntese , Animais , Toxinas Botulínicas/farmacologia , Cálcio/farmacologia , Células Cultivadas , Secreção de Insulina , Proteínas do Tecido Nervoso/efeitos dos fármacos , Ratos , Proteína 25 Associada a SinaptossomaRESUMO
The neural cell adhesion molecule L1 is a phosphorylated, integral membrane glycoprotein that is recovered from adult mouse brain tissue by immunoaffinity chromatography as a set of polypeptides with apparent molecular masses of 200, 180, 140, and 80 kilodaltons (L1-200, L1-180, L1-140, and L1-80, respectively). It has been shown that L1-140 and the phosphorylated L1-80 is generated from L1-200 by mild proteolytic treatment of intact cells. In the present study we have investigated the structural relationships between the different molecular forms of L1 and their location with regard to the surface membrane. We could show that L1-200 has two preferred cleavage sites, one that generates the amino terminal, extracellularly exposed L1-140 and the carboxy terminal L1-80 that spans the membrane. Cleavage at the other site leads to the generation of the amino terminally located L1-180 and the membrane-attached, phosphorylated carboxy terminal L1-30. This site is cleaved during treatment of live cultured cells with broad-spectrum, protease-free phospholipase C (but not phosphatidylinositol-specific phospholipase C) or exposure to sodium azide or cyanogen bromide. Other conditions that cause damage to cells do not lead to the generation of L1-180 and L1-30, suggesting a particular cell-intrinsic cleavage mechanism. L1-180 is truly soluble in aqueous solutions, since it can be recovered from culture supernatants and in the supernatant of a crude membrane fraction after incubation for 2 h at 37 degrees C. Although trypsin treatment alone does not release L1-140 into the supernatant, combination of phospholipase C and mild tryptic treatment leads to the release of L1-140 and L1-50, the latter being most likely the extracellularly exposed domain of L1-80 that is complementary to the membrane-integrated phosphorylated L1-30. Phase separation experiments with Triton X-114 show that the released forms of L1-180 and L1-140 distribute into the aqueous phase, whereas they distribute into the detergent phase when in association with L1-200 or L1-80. However, when L1-80 is cleaved to yield the soluble L1-50 and membrane-anchored L1-30, L1-140 is released into the supernatant together with L1-50. A strong affinity of L1-200, L1-140, and L1-80 to each other is also indicated by the fact that they incorporate together into liposomes and separate only under strong detergent conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Antígenos de Superfície/metabolismo , Encéfalo/metabolismo , Animais , Antígenos de Superfície/análise , Adesão Celular , Moléculas de Adesão Celular , Membrana Celular/análise , Membrana Celular/metabolismo , Cerebelo/análise , Cerebelo/metabolismo , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Imunoensaio , Técnicas de Imunoadsorção , Glicoproteínas de Membrana , Camundongos , Tripsina/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
To study the membrane anchoring of the 120 kDa component of the neural cell adhesion molecule N-CAM, the smallest form lacking a transmembrane domain, cultured mouse neural cells were treated with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. When live cultures of astrocytes and neurons are treated with phosphatidylinositol-specific phospholipase C, N-CAM120 is released into the supernatant. Under these conditions N-CAM140 and N-CAM180 are not released. Phospholipase C from Bacillus cereus or Clostridium perfringens does not release N-CAM120. The embryonic form of N-CAM on astrocytes migrating as a broad band between 120 and 180 kDa is also partially released by phosphatidylinositol-specific phospholipase C as a band migrating between 120 and 160 kDa. These observations suggest novel mechanisms in regulation of N-CAM120 expression on the cell surface and in modulation of N-CAM-mediated cell adhesion.