RESUMO
Mammalian spermatozoa employ calcium (Ca2+) and cyclic adenosine monophosphate (cAMP) signaling in generating flagellar beat. However, how sperm direct their movement towards the egg cells has remained elusive. Here we show that the Rho small G protein RAC1 plays an important role in controlling progressive motility, in particular average path velocity and linearity. Upon RAC1 inhibition of wild type sperm with the drug NSC23766, progressive movement is impaired. Moreover, sperm from mice homozygous for the genetically variant t-haplotype region (tw5/tw32), which are sterile, show strongly enhanced RAC1 activity in comparison to wild type (+/+) controls, and quickly become immotile in vitro. Sperm from heterozygous (t/+) males, on the other hand, display intermediate RAC1 activity, impaired progressive motility and transmission ratio distortion (TRD) in favor of t-sperm. We show that t/+-derived sperm consist of two subpopulations, highly progressive and less progressive. The majority of highly progressive sperm carry the t-haplotype, while most less progressive sperm contain the wild type (+) chromosome. Dosage-controlled RAC1 inhibition in t/+ sperm by NSC23766 rescues progressive movement of (+)-sperm in vitro, directly demonstrating that impairment of progressive motility in the latter is caused by enhanced RAC1 activity. The combined data show that RAC1 plays a pivotal role in controlling progressive motility in sperm, and that inappropriate, enhanced or reduced RAC1 activity interferes with sperm progressive movement. Differential RAC1 activity within a sperm population impairs the competitiveness of sperm cells expressing suboptimal RAC1 activity and thus their fertilization success, as demonstrated by t/+-derived sperm. In conjunction with t-haplotype triggered TRD, we propose that Rho GTPase signaling is essential for directing sperm towards the egg cells.
Assuntos
Aminoquinolinas/farmacologia , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/metabolismo , Pirimidinas/farmacologia , Motilidade dos Espermatozoides/genética , Motilidade dos Espermatozoides/fisiologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/metabolismo , Região do Complexo-t do Genoma/genética , Animais , Bovinos , Genótipo , Haplótipos , Heterozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/genética , Fenótipo , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
The t-haplotype, a variant form of the t-complex region on mouse chromosome 17, acts as selfish genetic element and is transmitted at high frequencies (> 95%) from heterozygous (t/+) males to their offspring. This phenotype is termed transmission ratio distortion (TRD) and is caused by the interaction of the t-complex responder (Tcr) with several quantitative trait loci (QTL), the t-complex distorters (Tcd1 to Tcd4), all located within the t-haplotype region. Current data suggest that the distorters collectively impair motility of all sperm derived from t/+ males; t-sperm is rescued by the responder, whereas (+)-sperm remains partially dysfunctional. Recently we have identified two distorters as regulators of RHO small G proteins. Here we show that the nucleoside diphosphate kinase gene Nme3 acts as a QTL on TRD. Reduction of the Nme3 dosage by gene targeting of the wild-type allele enhanced the transmission rate of the t-haplotype and phenocopied distorter function. Genetic and biochemical analysis showed that the t-allele of Nme3 harbors a mutation (P89S) that compromises enzymatic activity of the protein and genetically acts as a hypomorph. Transgenic overexpression of the Nme3 t-allele reduced t-haplotype transmission, proving it to be a distorter. We propose that the NME3 protein interacts with RHO signaling cascades to impair sperm motility through hyperactivation of SMOK, the wild-type form of the responder. This deleterious effect of the distorters is counter-balanced by the responder, SMOK(Tcr), a dominant-negative protein kinase exclusively expressed in t-sperm, thus permitting selfish behaviour and preferential transmission of the t-haplotype. In addition, the previously reported association of NME family members with RHO signaling in somatic cell motility and metastasis, in conjunction with our data involving RHO signaling in sperm motility, suggests a functional conservation between mechanisms for motility control in somatic cells and spermatozoa.
Assuntos
Nucleosídeo NM23 Difosfato Quinases/genética , Locos de Características Quantitativas , Motilidade dos Espermatozoides/genética , Proteínas rho de Ligação ao GTP , Região do Complexo-t do Genoma/genética , Sequência de Aminoácidos , Animais , Hereditariedade , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Fenótipo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Locos de Características Quantitativas/genética , Espermatozoides/fisiologia , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Lfc is a guanine nucleotide exchange factor (GEF) for Rho that demonstrates an unusual ability to associate with microtubules. While several phosphorylated residues have been detected in the Lfc polypeptide, the mechanism(s) by which phosphorylation regulates the exchange activity of Lfc remains unclear. We confirm that Lfc is a phosphorylated protein and demonstrate that 14-3-3 interacts directly and in a phosphorylation-dependent manner with Lfc. We identify AKAP121 as an Lfc-binding protein and show that Lfc is phosphorylated in an AKAP-dependent manner by protein kinase A (PKA). Forskolin treatment induced 14-3-3 binding to Lfc and suppressed the exchange activity of wild-type Lfc on RhoA. Importantly, a mutant of Lfc that is unable to associate with 14-3-3 proteins was resistant to inhibition by forskolin. Tctex-1, a dynein motor light chain, binds to Lfc in a competitive manner with 14-3-3.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas 14-3-3/metabolismo , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , Linhagem Celular , Sequência Consenso , Dineínas , Ativação Enzimática , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho , Fibras de Estresse/enzimologia , Proteína rhoA de Ligação ao GTP/metabolismo , Região do Complexo-t do GenomaRESUMO
The mechanisms that regulate symmetric, proliferative divisions versus asymmetric, neurogenic divisions of mammalian neural precursors are still not well understood. We found that Lfc (Arhgef2), a Rho-specific guanine nucleotide exchange factor that interacts with spindle microtubules, and its negative regulator Tctex-1 (Dynlt1) determine the genesis of neurons from precursors in the embryonic murine cortex. Specifically, genetic knockdown of Arhgef2 in cortical precursors either in culture or in vivo inhibited neurogenesis and maintained cells as cycling radial precursors. Conversely, genetic knockdown of Dynlt1 in radial precursors promoted neurogenesis and depleted cycling cortical precursors. Coincident silencing of these two genes indicated that Tctex-1 normally inhibits the genesis of neurons from radial precursors by antagonizing the proneurogenic actions of Lfc. Moreover, Lfc and Tctex-1 were required to determine the orientation of mitotic precursor cell divisions in vivo. Thus, Lfc and Tctex-1 interact to regulate cortical neurogenesis, potentially by regulating mitotic spindle orientation.
Assuntos
Córtex Cerebral/embriologia , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas Associadas aos Microtúbulos/genética , Neurogênese/fisiologia , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Células-Tronco/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Polaridade Celular/genética , Proliferação de Células , Células Cultivadas , Córtex Cerebral/citologia , Regulação para Baixo/genética , Dineínas , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Mitose/genética , Neurônios/citologia , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais/genética , Fuso Acromático/genética , Células-Tronco/citologia , Proteína rhoA de Ligação ao GTP/genética , Região do Complexo-t do GenomaRESUMO
Despite extensive data demonstrating that immature retroviral particle assembly can take place either at the plasma membrane or at a distinct location within the cytoplasm, targeting of viral precursor proteins to either assembly site still remains poorly understood. Biochemical data presented here suggest that Tctex-1, a light chain of the molecular motor dynein, is involved in the intracellular targeting of Mason-Pfizer monkey virus (M-PMV) polyproteins to the cytoplasmic assembly site. Comparison of the three-dimensional structures of M-PMV wild-type matrix protein (wt MA) with a single amino acid mutant (R55F), which redirects assembly from a cytoplasmic site to the plasma membrane, revealed different mutual orientations of their C- and N-terminal domains. This conformational change buries a putative intracellular targeting motif located between both domains in the hydrophobic pocket of the MA molecule, thereby preventing the interaction with cellular transport mechanisms.
Assuntos
Membrana Celular/metabolismo , Membrana Celular/virologia , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Nucleares/fisiologia , Retroviridae/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Células COS , Chlorocebus aethiops , Citoplasma/metabolismo , Humanos , Vírus dos Macacos de Mason-Pfizer/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fenótipo , Estrutura Terciária de Proteína , Região do Complexo-t do GenomaRESUMO
The mechanisms of axonal transport of the alphaherpesviruses, HSV and pseudorabies virus (PrV), in neuronal axons are of fundamental interest, particularly in comparison with other viruses, and offer potential sites for antiviral intervention or development of gene therapy vectors. These herpesviruses are transported rapidly along microtubules (MTs) in the retrograde direction from the axon terminus to the dorsal root ganglion and then anterogradely in the opposite direction. Retrograde transport follows fusion and deenvelopment of the viral capsid at the axonal membrane followed by loss of most of the tegument proteins and then binding of the capsid via one or more viral proteins (VPs) to the retrograde molecular motor dynein. The HSV capsid protein pUL35 has been shown to bind to the dynein light chain Tctex1 but is likely to be accompanied by additional dynein binding of an inner tegument protein. The mechanism of anterograde transport is much more controversial with different processes being claimed for PrV and HSV: separate transport of HSV capsid/tegument and glycoproteins versus PrV transport as an enveloped virion. The controversy has not been resolved despite application, in several laboratories, of confocal microscopy (CFM), real-time fluorescence with viruses dual labelled on capsid and glycoprotein, electron microscopy in situ and immuno-electron microscopy. Different processes for each virus seem counterintuitive although they are the most divergent in the alphaherpesvirus subfamily. Current hypotheses suggest that unenveloped HSV capsids complete assembly in the axonal growth cones and varicosities, whereas with PrV unenveloped capsids are only found travelling in a retrograde direction.
Assuntos
Herpes Simples/virologia , Neurônios/virologia , Simplexvirus/fisiologia , Animais , Axônios/virologia , Transporte Biológico , Proteínas do Capsídeo/metabolismo , Dineínas/metabolismo , Gânglios Espinais/virologia , Herpesvirus Suídeo 1/fisiologia , Humanos , Cinesinas/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Transporte Proteico , Replicação Viral , Região do Complexo-t do GenomaRESUMO
Tctex-1, a light-chain component of the cytoplasmic dynein motor complex, can function independently of dynein to regulate multiple steps in neuronal development. However, how dynein-associated and dynein-free pools of Tctex-1 are maintained in the cell is not known. Tctex-1 was recently identified as a Gbetagamma-binding protein and shown to be identical to the receptor-independent activator of G protein signaling AGS2. We propose a novel role for the interaction of Gbetagamma with Tctex-1 in neurite outgrowth. Ectopic expression of either Tctex-1 or Gbetagamma promotes neurite outgrowth whereas interfering with their function inhibits neuritogenesis. Using embryonic mouse brain extracts, we demonstrate an endogenous Gbetagamma-Tctex-1 complex and show that Gbetagamma co-segregates with dynein-free fractions of Tctex-1. Furthermore, Gbeta competes with the dynein intermediate chain for binding to Tctex-1, regulating assembly of Tctex-1 into the dynein motor complex. We propose that Tctex-1 is a novel effector of Gbetagamma, and that Gbetagamma-Tctex-1 complex plays a key role in the dynein-independent function of Tctex-1 in regulating neurite outgrowth in primary hippocampal neurons, most likely by modulating actin and microtubule dynamics.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Neuritos/fisiologia , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Células Cultivadas , Dineínas , Polarização de Fluorescência , Imunoprecipitação , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Neuritos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Região do Complexo-t do GenomaRESUMO
Genetic transformation of plants by Agrobacterium, which in nature causes neoplastic growths, represents the only known case of trans-kingdom DNA transfer. Furthermore, under laboratory conditions, Agrobacterium can also transform a wide range of other eukaryotic species, from fungi to sea urchins to human cells. How can the Agrobacterium virulence machinery function in such a variety of evolutionarily distant and diverse species? The answer to this question lies in the ability of Agrobacterium to hijack fundamental cellular processes which are shared by most eukaryotic organisms. Our knowledge of these host cellular functions is critical for understanding the molecular mechanisms that underlie genetic transformation of eukaryotic cells. This review outlines the bacterial virulence machinery and provides a detailed discussion of seven major biological systems of the host cell-cell surface receptor arrays, cellular motors, nuclear import, chromatin targeting, targeted proteolysis, DNA repair, and plant immunity--thought to participate in the Agrobacterium-mediated genetic transformation.
Assuntos
Plantas/genética , Rhizobium/genética , Rhizobium/patogenicidade , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Reparo do DNA , Regulação da Expressão Gênica de Plantas , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Plantas/imunologia , Plantas/microbiologia , Transporte Proteico , Receptores de Superfície Celular/metabolismo , Transformação Genética , Virulência , Região do Complexo-t do GenomaRESUMO
G protein-coupled receptors (GPCRs) are seven transmembrane receptors with an N-terminus in the extracellular region and C-terminus in the intracellular region. When an agonist binds to a GPCR, a signal is transduced into a cell through the activation of trimeric G proteins. Recently, it has been shown that the activities of GPCRs are regulated by multiple mechanisms. One of the mechanisms is regulation through the binding proteins to the carboxy (C)-terminus of GPCRs. In the present study, the binding partners for the C-terminus of the parathyroid hormone receptor (PTHR) and thromboxane A(2) receptor (TP) were searched for using yeast two-hybrid screening, and the functions of these proteins were investigated. We identified t-complex testis expressed-1 (Tctex-1) and 4.1G as associated proteins for the PTHR. Tctex-1 is one of the light chains of cytoplasmic dynein, which is a motor protein across microtubles. We found that Tctex-1 was involved in agonist-induced internalization of the PTHR. 4.1G, a cytoskeletal protein, facilitated the cell surface localization of the PTHR and augmented PHTR-mediated signal transduction. TPs consists of two splicing variants, TPalpha and TPbeta. As a result of yeast two-hybrid screening, two proteasomal proteins, proteasome activator PA28gamma and proteasome subunit alpha7, were identified as direct interacting proteins for TPbeta. TPbeta has a tendency to be retained in the intracellular compartment, probably due to its binding to proteasomes. We also demonstrated that TPalpha and TPbeta formed heterodimers, and the signal transduction through TPalpha was reduced by the formation of heterodimers. In conclusion, the proteins bound to GPCRs may regulate the intracellular traffic of GPCRs.
Assuntos
Receptor Tipo 1 de Hormônio Paratireóideo/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Tromboxano A2 e Prostaglandina H2/fisiologia , Animais , Autoantígenos/fisiologia , Proteínas do Citoesqueleto/fisiologia , Dineínas , Proteínas de Ligação ao GTP/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Nucleares/fisiologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Ligação Proteica , Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptores Acoplados a Proteínas G/química , Transdução de Sinais , Região do Complexo-t do GenomaRESUMO
Endoglin is a membrane-inserted protein that is preferentially synthesized in angiogenic vascular endothelial and smooth muscle cells. Endoglin associates with members of the transforming growth factor-beta (TGF-beta) receptor family and has been identified as the gene involved in hereditary hemorrhagic telangiectasia. Although endoglin is known to affect cell responses to TGF-beta, its mode of action is largely unknown. We performed yeast two-hybrid screening of a human placental cDNA library and isolated a new endoglin-binding partner, a novel 221-amino acid member of the Tctex1/2 family of cytoplasmic dynein light chains named Tctex2beta, as the founder of a new Tctex1/2 subfamily. The interaction was localized exclusively to the cytoplasmic domain of endoglin. Reverse transcription-PCR showed expression of Tctex2beta in a wide range of tissues, including vascular endothelial and smooth muscle cells, placenta, and testis, as well as in several tumor cell lines. High expression levels were found in human umbilical vein endothelial cells and the large cell lung cancer cell line. Forced expression of Tctex2beta had a profound inhibitory effect on TGF-beta signaling. Additional Tctex2beta-interacting receptors were identified to be the TGF-beta type II receptor and most likely beta-glycan, but not ALK5, ALK1, or the bone morphogenetic protein type II receptor. Upon fluorescence tagging, co-localization of Tctex2beta and endoglin, as well as Tctex2beta, endoglin, and the TGF-beta type II receptor, was observed by different microscopy techniques. Our findings link endoglin for the first time to microtubule-based minus end-directed transport machinery, suggesting that some endoglin functions might be regulated and directed by its interaction with the cytoplasmic dynein light chain Tctex2beta.
Assuntos
Proteínas de Transporte/química , Proteínas de Drosophila/química , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Nucleares/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Células COS , Chlorocebus aethiops , Dineínas , Humanos , Camundongos , Vison , Dados de Sequência Molecular , Células NIH 3T3 , Filogenia , Ligação Proteica , Homologia de Sequência de Aminoácidos , Região do Complexo-t do GenomaRESUMO
Post-Golgi to apical surface delivery in polarized epithelial cells requires the cytoplasmic dynein motor complex. However, the nature of dynein-cargo interactions and their underlying regulation are largely unknown. Previous studies have shown that the apical surface targeting of rhodopsin requires the dynein light chain, Tctex-1, which binds directly to both dynein intermediate chain (IC) and rhodopsin. In this report, we show that the S82E mutant of Tctex-1, which mimics Tctex-1 phosphorylated at serine 82, has a reduced affinity for dynein IC but not for rhodopsin. Velocity sedimentation experiments further suggest that S82E is not incorporated into the dynein complex. The dominant-negative effect of S82E causes rhodopsin mislocalization in polarized Madin-Darby canine kidney (MDCK) cells. The S82A mutant, which mimics dephosphorylated Tctex-1, can be incorporated into dynein complex but is impaired in its release. Expression of S82A also causes disruption of the apical localization of rhodopsin in MDCK cells. Taken together, these results suggest that the dynein complex disassembles to release cargo due to the specific phosphorylation of Tctex-1 at the S82 residue and that this process is critical for the apical delivery of membrane cargoes.
Assuntos
Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Rodopsina/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Contactina 1 , Cães , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Rodopsina/genética , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Taninos/farmacologia , Junções Íntimas/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Proteína da Zônula de Oclusão-1 , Região do Complexo-t do GenomaRESUMO
Cell-substrate contacts, called focal adhesions (FAs), are dynamic in rapidly moving cells. We show that supervillin (SV)--a peripheral membrane protein that binds myosin II and F-actin in such cells--negatively regulates stress fibers, FAs, and cell-substrate adhesion. The major FA regulatory sequence within SV (SV342-571) binds to the LIM domains of two proteins in the zyxin family, thyroid receptor-interacting protein 6 (TRIP6) and lipoma-preferred partner (LPP), but not to zyxin itself. SV and TRIP6 colocalize within large FAs, where TRIP6 may help recruit SV. RNAi-mediated decreases in either protein increase cell adhesion to fibronectin. TRIP6 partially rescues SV effects on stress fibers and FAs, apparently by mislocating SV away from FAs. Thus, SV interactions with TRIP6 at FAs promote loss of FA structure and function. SV and TRIP6 binding partners suggest several specific mechanisms through which the SV-TRIP6 interaction may regulate FA maturation and/or disassembly.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adesões Focais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fatores de Transcrição/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Células COS , Bovinos , Células Cultivadas , Chlorocebus aethiops , Regulação para Baixo/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas com Domínio LIM , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos de Músculo Liso/citologia , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Ratos , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/química , Região do Complexo-t do GenomaRESUMO
The identity and biology of stem cells and progenitors in the adult brain are of considerable interest, because these cells hold great promise for the development of novel therapies for damaged brain tissue in human diseases. This research field critically needs biological markers that specifically identify the resident precursors in the germinal zones of the adult central nervous system so that the discovery of regulatory influences for adult neurogenesis may be facilitated. In this study, by using a combination of in situ hybridization, bromodeoxyuridine incorporation, immunocolocalization, and ultrastructural studies, we show that in rodents Tctex-1, a cytoplasmic dynein light chain, is selectively enriched in almost all cycling progenitors and young neuronal progeny, but not in mature granular cells and astrocytes, in the subgranular zone of the adult dentate gyrus. Tctex-1 is also selectively abundant in cells closely resembling previously described immature progenitors and migrating neuroblasts at the subventricular zone of the lateral ventricle. Our results suggest that Tctex-1 serves as a novel marker for the identification of neural progenitors of the adult brain.
Assuntos
Astrócitos/metabolismo , Giro Denteado/citologia , Giro Denteado/metabolismo , Proteínas Associadas aos Microtúbulos/biossíntese , Neurônios/metabolismo , Proteínas Nucleares/biossíntese , Células-Tronco/metabolismo , Animais , Biomarcadores/análise , Bromodesoxiuridina , Giro Denteado/ultraestrutura , Proteínas do Domínio Duplacortina , Dineínas , Feminino , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/biossíntese , Neuropeptídeos/biossíntese , Proteínas de Ligação a RNA/biossíntese , Ratos , Ratos Sprague-Dawley , Região do Complexo-t do GenomaRESUMO
Recombinant expression of actin in bacteria results in non-native species that aggregate into inclusion bodies. Actin is a folding substrate of TRiC, the chaperonin of the eukaryotic cytosol. By employing bacterial in vitro translation lysates supplemented with purified chaperones, we have found that TRiC is the only eukaryotic chaperone necessary for correct folding of newly translated actin. The actin thus produced binds deoxyribonuclease I and polymerizes into filaments, hallmarks of its native state. In contrast to its rapid folding in the eukaryotic cytosol, actin translated in TRiC-supplemented bacterial lysate folds with slower kinetics, resembling the kinetics upon refolding from denaturant. Lysate supplementation with the bacterial chaperonin GroEL/ES or the DnaK/DnaJ/GrpE chaperones leads to prevention of actin aggregation, yet fails to support its correct folding. This combination of in vitro bacterial translation and TRiC-assisted folding allows a detailed analysis of the mechanisms necessary for efficient actin folding in vivo. In addition, it provides a robust alternative for the production of substantial amounts of eukaryotic proteins that otherwise misfold or lead to cellular toxicity upon expression in heterologous hosts.
Assuntos
Actinas/metabolismo , Bactérias/metabolismo , Chaperoninas/metabolismo , Células Eucarióticas/metabolismo , Biossíntese de Proteínas , Actinas/genética , Extratos Celulares , Chaperonina 10/genética , Chaperonina 10/metabolismo , Chaperonina com TCP-1 , Chaperoninas/genética , Citosol/metabolismo , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Cinética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Região do Complexo-t do GenomaRESUMO
Heterozygosity for a t haplotype (t) in male mice results in distorted transmission (TRD) of the t-bearing chromosome 17 homolog to their offspring. However, homozygosity for t causes male sterility, thus limiting the spread of t through the population at large. The Ca(2+)-dependent sperm tail curvature phenotypes, "fishhook", where abnormally high levels of sperm exhibit sharp bends in the midpiece, and "curlicue", where motile sperm exhibit a chronic negative curving of the entire tail, have been tightly linked to t-associated male TRD and sterility traits, respectively. Genetic studies have indicated that homozygosity for the t allele of Dnahc8, an axonemal gamma-type dynein heavy chain (gammaDHC) gene, is partially responsible for expression of "curlicue"; however, its involvement in "fishhook"/TRD, if any, is unknown. Here we report that the major isoform of DNAHC8 is copiously expressed, carries an extended N-terminus and full-length C-terminus, and is stable and equally abundant in both testis and sperm from +/+ and t/t animals. By in silico analysis we also demonstrate that at least three of the seventeen DNAHC8(t) mutations at highly conserved positions in wild-type DHCs may be capable of substantially altering normal DNAHC8 function. Interestingly, DNAHC8 is confined to the principal piece of the sperm tail. The combined results of this study suggest possible mechanisms of DNAHC8(t) dysfunction and involvement in "curlicue", and support the hypothesis that "curlicue" is a multigenic phenomenon. They also demonstrate that the accelerated "fishhook" phenotype of sperm from +/t males is not directly linked to DNAHC8(t) dysfunction.
Assuntos
Dineínas/química , Dineínas/genética , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Cauda do Espermatozoide/metabolismo , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Animais , Dineínas do Axonema , Sequência de Bases , DNA Complementar/genética , Dineínas/metabolismo , Haplótipos , Heterozigoto , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fenótipo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Cauda do Espermatozoide/ultraestrutura , Região do Complexo-t do GenomaRESUMO
Retroviral tagging has been used extensively and successfully to identify genes implicated in cancer pathways. In order to find oncogenes implicated in T-cell leukemia, we used the highly leukemogenic radiation leukemia retrovirus VL3 (RadLV/VL3). We applied the inverted PCR technique to isolate and analyze sequences flanking proviral integrations in RadLV/VL3-induced T lymphomas. We found retroviral integrations in c-myc and Pim1 as already reported but we also identified for the first time Notch1 as a RadLV common integration site. More interestingly, we found a new RadLV common integration site that is situated on mouse chromosome X (XA4 region, bp 45091000). This site has also been reported as an SL3-3 and Moloney murine leukemia virus integration site, which strengthens its implication in murine leukemia virus-induced T lymphomas. This locus, named Kis2 (Kaplan Integration Site 2), was found rearranged in 11% of the tumors analyzed. In this article, we report not only the alteration of the Kis2 gene located nearby in response to RadLV integration but also the induction of the expression of Phf6, situated about 250 kbp from the integration site. The Kis2 gene encodes five different alternatively spliced noncoding RNAs and the Phf6 gene codes for a 365-amino-acid protein which contains two plant homology domain fingers, recently implicated in the Börjeson-Forssman-Lehmann syndrome in humans. With the recent release of the mouse genome sequence, high-throughput retroviral tagging emerges as a powerful tool in the quest for oncogenes. It also allows the analysis of large DNA regions surrounding the integration locus.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , RNA não Traduzido/metabolismo , RNA Viral/metabolismo , Vírus da Leucemia Induzida por Radiação/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Leucemia Experimental/virologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Células NIH 3T3 , Proteínas Nucleares/genética , RNA Mensageiro/genética , RNA Viral/genética , Receptor Notch1 , Receptores de Superfície Celular/genética , Infecções por Retroviridae/virologia , Alinhamento de Sequência , Fatores de Transcrição/genética , Infecções Tumorais por Vírus/virologia , Integração Viral , Cromossomo X/genética , Região do Complexo-t do GenomaRESUMO
Coordinated microtubule and microfilament changes are essential for the morphological development of neurons; however, little is know about the underlying molecular machinery linking these two cytoskeletal systems. Similarly, the indispensable role of RhoGTPase family proteins has been demonstrated, but it is unknown how their activities are specifically regulated in different neurites. In this paper, we show that the cytoplasmic dynein light chain Tctex-1 plays a key role in multiple steps of hippocampal neuron development, including initial neurite sprouting, axon specification, and later dendritic elaboration. The neuritogenic effects elicited by Tctex-1 are independent from its cargo adaptor role for dynein motor transport. Finally, our data suggest that the selective high level of Tctex-1 at the growth cone of growing axons drives fast neurite extension by modulating actin dynamics and also Rac1 activity.
Assuntos
Actinas/metabolismo , Axônios/metabolismo , Dineínas/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas rac1 de Ligação ao GTP/fisiologia , Animais , Axônios/ultraestrutura , Polaridade Celular , Células Cultivadas , Clonagem Molecular , Cones de Crescimento/metabolismo , Hipocampo/citologia , Neuritos/fisiologia , Ratos , Região do Complexo-t do GenomaRESUMO
Most organisms use crossovers (chiasmata) to maintain physical connections between homologous chromosomes that ensure their proper segregation at the first meiotic division. The fission yeast Schizosaccharomyces pombe has a residual ability to segregate homologous chromosomes in the absence of meiotic recombination (achiasmate segregation). Using cytologically tagged chromosomes, we established a role for the microtubule motor dynein in meiotic chromosome segregation. Dhc1, the motor subunit of dynein, is required for chromosome segregation in both the presence and the absence of recombination. Dlc1, a member of the Tctex-1 dynein light-chain family, preferentially affects the segregation of achiasmate chromosomes. Dlc1 is the first identified protein, outside of Drosophila, that preferentially affects achiasmate chromosome segregation. We discuss possible roles of the dynein motor in this process.
Assuntos
Dineínas/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/fisiologia , Aneuploidia , Cromátides/metabolismo , Segregação de Cromossomos , Dineínas/química , Proteínas Fúngicas/metabolismo , Genótipo , Meiose , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Modelos Genéticos , Mutação , Proteínas Nucleares/metabolismo , Recombinação Genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Fuso Acromático , Região do Complexo-t do GenomaRESUMO
Voltage-gated Ca(2+) channels (VGCCs) are important in regulating a variety of cellular functions in neurons. It remains poorly understood how VGCCs with different functions are sorted within neurons. Here we show that the t-complex testis-expressed 1 (tctex1) protein, a light-chain subunit of the dynein motor complex, interacts directly and selectively with N- and P/Q-type Ca(2+) channels, but not L-type Ca(2+) channels. The interaction is insensitive to Ca(2+). Overexpression in hippocampal neurons of a channel fragment containing the binding domain for tctex1 significantly decreases the surface expression of endogenous N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels, as determined by immunostaining. Furthermore, disruption of the tctex1-Ca(2+) channel interaction significantly reduces the Ca(2+) current density in hippocampal neurons. These results underscore the importance of the specific tctex1-channel interaction in determining sorting and trafficking of neuronal Ca(2+) channels with different functionalities.
Assuntos
Canais de Cálcio/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Animais , Western Blotting/métodos , Cálcio/farmacologia , Canais de Cálcio/química , Canais de Cálcio/classificação , Carbodi-Imidas/metabolismo , Carbodi-Imidas/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Dineínas , Capacitância Elétrica , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Ativação do Canal Iônico , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Dados de Sequência Molecular , Mutagênese/fisiologia , Técnicas de Patch-Clamp/métodos , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Transfecção/métodos , Técnicas do Sistema de Duplo-Híbrido , Região do Complexo-t do GenomaRESUMO
Recent work from the King lab (Wu et al., 2005) on the structure of the Tctex1 dynein light chain provides new insights into the mechanism of cytoplasmic dynein cargo binding and the functional significance of light chain isoform diversity.