RESUMO
In the present contribution we report on a novel route to synthesize 2D-polyaniline (2D-PAN) on sulfonated-poly(styrene) (SPS) templates by allowing first monomer assembly followed by chemical oxidation to achieve polymerization. We show that Aplysia neurons grown on 2D-PAN exhibit an unusual growth pattern and adhesion to this conducting substrate that is manifested by the formation of giant lamellipodia. The lamellipodial domains are characterized by small gap between the plasma membrane and the 2D-PAN substrate (ca. 30 nm) and actin rich skeleton resembling the skeleton of growth cones. This behavior is characteristic to uniform substrates containing only 2D-PAN. However, in patterned substrates containing additionally poly(L-lysine) Aplysia neurons prefer to extend new neurites on the poly(L-lysine) domains.
Assuntos
Compostos de Anilina/química , Compostos de Anilina/farmacologia , Técnicas de Cultura de Células/métodos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Actinas/metabolismo , Compostos de Anilina/síntese química , Animais , Aplysia , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Células Cultivadas , Simulação por Computador , Condutividade Elétrica , Microscopia de Força Atômica , Microscopia Confocal , Microscopia Eletrônica , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Estrutura Molecular , Neurônios/metabolismo , Polilisina/metabolismo , Análise Espectral , Eletricidade Estática , Enxofre/químicaRESUMO
The green fluorescent fusion protein and its isoforms are extensively used to monitor gene expression, protein localisation and their dynamics in relations to fundamental cellular processes. However, it has not yet been effectively applied to Aplysia neurons that serve as a powerful model to study the mechanisms underlying neuroplasticity. We report here the development of a procedure combining in vitro transcription of mRNA encoding fluorescent-tagged proteins and its subsequent injection into the cytoplasm to image, in real-time, protein dynamics in cultured Aplysia neurones. To illustrate the efficiency of the procedure we report here the visualisation of actin, microtubules and vesicle trafficking. The results presented here introduce a reliable and effective method to express green fluorescent protein (GFP) fusion proteins in cultured Aplysia neurons.