RESUMO
Echinoderm microtubule-associated protein (EMAP) is the major microtubule binding protein in dividing sea urchin (Strongylocentrotus purpuratus) eggs. Echinoderm microtubule-associated protein like protein 4 (Eml4, restrictedly overexpressed proliferation-associated protein 120 kDa (Ropp120)) is one of the five mammalian EMAP homologues, the cellular function of which remains to be elucidated. In our first set of experiments we determined the spatio-temporal expression pattern of Eml4 in mouse brain. Our results demonstrate that Eml4 is a highly developmentally regulated gene with high expression levels in the developing nervous system of E11 embryos declining to low levels in adult. Spatially, Eml4 expression becomes restricted to the olfactory bulb, hippocampus and cerebellum. Transient transfection of a fusion construct of full-length mouse Eml4 with green fluorescent protein (GFP-Eml4) into Cos7 and HeLa cells resulted in colocalization of GFP-Eml4 with microtubules. This colocalization was observed both with microtubules of non-dividing cells and with the mitotic spindle of dividing cells. In addition, transient overexpression of GFP-Eml4 in Cos7 cells resulted in microtubules that were resistant to nocodazole treatment suggesting that Eml4 stabilizes microtubules. A consequence of microtubule stabilization is a net reduction in the amount of free tubulin. Microtubule stabilizing proteins therefore are expected to indirectly decrease the microtubule growth rate. Indeed, transient transfection of GFP-Eml4 resulted in a marked decrease in the microtubule growth rate, which is in line with our hypothesis that Eml4 functions as a microtubule stabilizing protein. In summary, our results suggest that Eml4 is a developmentally regulated protein that colocalizes with and stabilizes microtubules.
Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/ultraestrutura , Células COS , Chlorocebus aethiops , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/ultraestrutura , Nocodazol/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismo , Transfecção , Tubulina (Proteína)/metabolismoRESUMO
While firing rate is well established as a relevant parameter for encoding information exchanged between neurons, the significance of other parameters is more conjectural. Here, we show that regularity of neuronal spike activities affects sensorimotor processing in tottering mutants, which suffer from a mutation in P/Q-type voltage-gated calcium channels. While the modulation amplitude of the simple spike firing rate of their floccular Purkinje cells during optokinetic stimulation is indistinguishable from that of wild-types, the regularity of their firing is markedly disrupted. The gain and phase values of tottering's compensatory eye movements are indistinguishable from those of flocculectomized wild-types or from totterings with the flocculus treated with P/Q-type calcium channel blockers. Moreover, normal eye movements can be evoked in tottering when the flocculus is electrically stimulated with regular spike trains mimicking the firing pattern of normal simple spikes. This study demonstrates the importance of regularity of firing in Purkinje cells for neuronal information processing.
Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio Tipo P/genética , Córtex Cerebelar/fisiologia , Movimentos Oculares/fisiologia , Células de Purkinje/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Artefatos , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo P/efeitos dos fármacos , Córtex Cerebelar/citologia , Córtex Cerebelar/efeitos dos fármacos , Estimulação Elétrica , Retroalimentação/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Nistagmo Optocinético/fisiologia , Núcleo Olivar/fisiologia , Estimulação Luminosa , Células de Purkinje/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Núcleos Vestibulares/fisiologia , Percepção Visual/fisiologiaRESUMO
The GATA-3 transcription factor shows a specific and restricted expression pattern in the developing and adult mouse brain. In the present study we investigated the role of GATA-3 in the caudal raphe system, which is known to operate as a modulator of motor activity. We demonstrate that virtually all neurons in the caudal raphe nuclei that express GATA-3 also produce serotonin. Absence of GATA-3, as analyzed in chimeric -/- mice, affects the cytoarchitecture of serotonergic neurons in the caudal raphe nuclei. As a result the chimeras show a serious defect in their locomotor performance on a rotating rod. In sum, we conclude that GATA-3 plays a major role in the development of the serotonergic neurons of the caudal raphe nuclei, and that it is crucial for their role in locomotion.