RESUMO
To study the molecular basis for organized pigment granule transport, procedures were developed to lyse melanophores of Tilapia mossambica under conditions in which pigment granule movements could be reactivated. Gentle lysis of the melanophores resulted in a permeabilized cell model, which, in the absence of exogenous ATP, could undergo multiple rounds of pigment granule aggregation and dispersion when sequentially challenged with epinephrine and cAMP. Both directions of transport required ATP, since aggregation or dispersion in melanophores depleted of nucleotides could be reactivated only upon addition of MgATP or MgATP plus cAMP, respectively. Differences between the nucleotide sensitivities for aggregation and dispersion were demonstrated by observations that aggregation had a lower apparent Km for ATP than did dispersion and could be initiated at a lower ATP concentration. Moreover, aggregation could be initiated by ADP, but only dispersion could be reactivated by the thiophosphate ATP analog, ATP gamma S. The direction of pigment transport was determined solely by cAMP, since pigment granules undergoing dispersion reaggregated when cAMP was removed, and those undergoing aggregation dispersed when cAMP was added. These results provide evidence that pigment granule motility may be based on two distinct mechanisms that are differentially activated and regulated to produce bidirectional movements.
Assuntos
Melanóforos/fisiologia , Microtúbulos/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , AMP Cíclico/metabolismo , Epinefrina/farmacologia , Peixes , Movimento , Nucleotídeos/metabolismoRESUMO
Microtubule (MT)-binding peptides have been detected in homogenates of bovine brain tissue utilizing a blot overlay assay. Blots were prepared by the electrophoretic transfer to nitrocellulose of proteins separated on polyacrylamide gels. These blots were incubated with taxol stabilized MTs or tubulin, rinsed, and then fixed by air drying. About 17 soluble MT-associated proteins (MAPs) were identified by immunodetection of bound tubulin, including MAP2, kinesin, and tau. The interaction of MTs with these peptides appears to be specific, since MT binding can be displaced by a fluorescent tubulin analog, is competitively inhibited by the addition of exogenous brain MAPs, is decreased by raising the salt concentration, and is diminished by sodium dodecyl sulfate (SDS) denaturation. Only one protein (150 kDa) appears to have an interaction with MTs that is stable in high salt. The specificity of the binding on blots is further illustrated by the interaction of MTs with the MT-binding domains of MAP2 (32-35 kDa fragments) and kinesin (64 kDa fragment). Specific MT-binding peptides or domains can thus be isolated and characterized with this method, which requires little protein and is suitable for use with proteins that are either soluble or insoluble under physiological conditions.
Assuntos
Química Encefálica , Proteínas Associadas aos Microtúbulos/análise , Microtúbulos/metabolismo , Animais , Bovinos , Concentração de Íons de Hidrogênio , Immunoblotting , Proteínas Associadas aos Microtúbulos/metabolismo , Mapeamento de Peptídeos , Tubulina (Proteína)/metabolismoRESUMO
The development of procedures to lyse and reactivate pigment granule movements in chromatophores has provided the only information to date concerning the mechanisms by which cells regulate the direction of organelle transport. Continued analysis of motility in these models as well as in a reconstituted system containing only the pigment granules, the appropriate cytoskeletal structures, and defined soluble cell components should contribute to our understanding of the mechanisms by which protein phosphorylation and dephosphorylation or Ca2+ regulate direction of transport and to the identification and characterization of the force-generating proteins responsible for producing bidirectional organelle movements.
Assuntos
Cromatóforos/ultraestrutura , Organelas/ultraestrutura , Animais , Fracionamento Celular/métodos , Cromatóforos/citologia , Cromatóforos/fisiologia , Modelos Biológicos , Organelas/fisiologia , Percas , Pele/citologiaRESUMO
Studies were conducted to investigate the molecular basis for bidirectional pigment granule transport in digitonin-lysed melanophores. Pigment granule dispersion, but not aggregation, required cAMP and resulted in the phosphorylation of a 57 kd polypeptide. cAMP-dependent protein kinase inhibitor prevented this phosphorylation as well as pigment dispersal. In contrast, both pigment aggregation and the concomitant dephosphorylation of the 57 kd polypeptide were blocked by phosphatase inhibitors. These data support a model in which pigment dispersion and aggregation require protein phosphorylation and dephosphorylation, respectively. Furthermore, studies using the ATP analog, ATP gamma S, suggest either that protein phosphorylation alone is sufficient for dispersion or that transport is mediated by a unique force-generating ATPase that can use ATP gamma S for hydrolyzable energy.
Assuntos
Melanóforos/metabolismo , Pigmentos Biológicos/metabolismo , Proteínas/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , AMP Cíclico/farmacologia , Melanóforos/ultraestrutura , Modelos Biológicos , Percas , Fosforilação , Proteínas Quinases/metabolismoRESUMO
The multichain T cell antigen receptor (TCR) is composed of an antigen binding (alpha/beta) domain and associated signal-transducing complexes, the CD3 (gamma, delta, and epsilon) and the zeta chains. The zeta chain (TCR zeta) plays a key role in signal transduction. We show here that TCR ligation induces association of tyrosine-phosphorylated TCR zeta with the detergent-insoluble cell fraction. The microfilament poison, cytochalasin D, disrupts this association and enhances the coprecipitation of actin with TCR zeta after receptor ligation. This microfilament association is specific to TCR-associated polypeptides containing at least one intact immunoreceptor tyrosine-based activation motif (ITAM). Mapping studies using transfectants and chimeric TCR zeta chain constructs suggest that the third ITAM is necessary and sufficient for association, if the distal tyrosine is intact. This cytoskeletal association is directly correlated with IL-2 production, and ligation of TCR on immature thymocytes does not induce TCR zeta-cytoskeleton association. These data thus provide direct evidence of a developmentally regulated activation-dependent interaction between a lymphocyte antigen receptor and the actin cytoskeleton.
Assuntos
Actinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Proteínas do Citoesqueleto/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Fosforilação , Testes de Precipitina , Transdução de Sinais/fisiologiaRESUMO
The TCR zeta-chain (zeta) on mature murine T lymphocytes binds to the microfilament cytoskeleton in response to Ag receptor ligation. Here, we report the role of Src family kinases in zeta-cytoskeletal binding, using mutant mice and a cell-free model system. Binding of zeta to actin in the cell-free system has a specific requirement for ATP and divalent cations, with an apparent Michaelis-Menton constant for ATP in the millimolar range, and can be disrupted by either EDTA or the microfilament poison, cytochalasin D, suggesting that microfilaments provide the structural framework for an active process involving cellular kinases. Indeed, tyrosine-phosphorylated zeta is a predominant form of the zeta-chain bound to polymerized actin, while challenge with alkaline phosphatase prevents zeta-chain association in solution and releases zeta-chain from the bound state. Phosphorylated Src-family kinase pp56Lck also associates with membrane skeleton upon TCR engagement and is a component of the reconstituted cytoskeletal pellet. Zeta-chain phosphorylation and zeta-cytoskeletal binding are abrogated in cell lysates with reduced levels of pp56Lck and in activated mutant murine T cells lacking pp56Lck, implicating pp56Lck as the kinase involved in zeta-chain tyrosine phosphorylation and zeta-cytoskeletal binding. Finally, recombinant Lck Src homology 2 domain preferentially inhibits reconstituted zeta-cytoskeleton association, suggesting that zeta-microfilament binding is dependent on interactions between phosphorylated tyrosine residues in zeta-chain activation motifs and the Src homology 2 domain of the Lck protein tyrosine kinase.
Assuntos
Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Citoesqueleto de Actina/enzimologia , Citoesqueleto de Actina/imunologia , Citoesqueleto de Actina/metabolismo , Trifosfato de Adenosina/fisiologia , Animais , Cátions Bivalentes , Sistema Livre de Células , Citoesqueleto/enzimologia , Ativação Linfocitária/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Knockout , Fosforilação , Ligação Proteica/imunologia , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Linfócitos T/enzimologia , Linfócitos T/imunologia , Tirosina/metabolismo , Domínios de Homologia de src/imunologia , Quinases da Família srcRESUMO
The zeta subunit of the TCR complex targets receptor surface expression, is phosphorylated on tyrosine residues upon T cell activation, and is implicated in signal transduction after TCR ligation. Here we show that, although intrathymic expression of the murine TCR-associated zeta-chain relative to the other chains of the Ag receptor complex remains unchanged during early thymocyte development, there is a doubling of TCR-associated zeta-chain surface expression upon thymocyte maturation. The ratio of tyrosine-phosphorylated relative to nonphosphorylated TCR-associated zeta-chain also changes with thymocyte development. This ratio was quantified after the purification and detergent extraction of receptor complexes from freshly isolated immature or mature thymocytes. Immunoprecipitation of the zeta-chain released from the complex allowed for the isolation of the tyrosine-phosphorylated and nonphosphorylated forms of TCR-associated zeta-chain. Intracellular free zeta-chain was characterized by immunoprecipitation after clearing the cell lysate of intact TCR complexes. Densitometric analysis of immunoblots indicated that surface phosphorylated zeta-chain is more abundant in immature relative to mature T cell populations, whereas the inverse is true of intracellular phosphorylated zeta-chain. Surface phosphorylated zeta-chain also migrated at a higher m.w. than its cytoplasmic counterpart, suggesting that it is more highly modified on some or all of its available tyrosines. These findings demonstrate that the stoichiometry and post-translational modification of the TCR complex are regulated, in vivo, and may determine the functional maturation of T cell signaling, selection, and activation.