Evidence for the involvement of SARA in endosomal trafficking and neuronal polarization

ARIAS CI; SUNG CH; CÁCERES A; CONDE C

Buzios, Brasil

Congreso; I Congreso IBRO/LARC de Neurociencias de América Latina, Caribe y Península Ibérica; 2008

IBRO/LARC

SARA is a protein, which has a binding domain to Smad2 and Smad3 proteins and a double zinc-finger domain FYVE (Fab1p, YOTB, Vac1p y EEA1) that enables it to attach to PI3P with high specificity and to localize to early endosomes. It has been demonstrated in cell lines that SARA plays a crucial role in the Rab5-regulated endosomal trafficking. Since membrane recycling is an essential event in the establishment of neuronal polarity, in this study we have analyzed the function of SARA in the regulation of process formation and membrane delivery in cultured hippocampal pyramidal neurons. Confocal microscopy revealed that SARA is endogenously expressed in hippocampal neurons and partially colocalize with Rab5. The overexpression of SARA causes an enlargement of the early endosomal compartment labelled with Rab5-GFP. The cells coexpressing SARA and Rab11-GFP were unable to form a typical recycling endosome as evidenced by the lack of the yuxtanuclear Rab11 positive structure. The suppression of SARA using siRNA and cotransfected with Rab11GFP display a large recycling endosome. Morphometric analysis of neurons ectopically expressing SARA revealed a significantly reduction in the total neuritic length; by contrast, SARA suppression induced neurons with multiple and large axons compared with GFP control cells. These observations prompted us to analyse with more detail the participation of SARA in membrane addition. L1 is a neuronal adhesion molecule that is important for axon outgrowth and pathfinding, being constantly endocytosed and recycled back to the plasma membrane in the axonal growth cone. Neurons overexpressing SARA shown less L1 at the axonal membrane compared with control ones, while those transfected with SARA siRNA showed an enhanced membrane delivery. In support of this, TIRF microscopy revealed that neurons co-expressing SARA and the transferrin receptor fused to GFP (TfRGFP) showed a significant reduction of fusion events at the somatodendritic plasma membrane. Taken together, our results suggest that SARA regulates neuronal polarity by regulating the trafficking of membrane from the early endosome to the recycling endosome, both in axons and dendrites.