hat such amino acid collaborates in the NES2 binding to CRM1 and enhances NES function. In the case of full length Ngn3, the point mutation L135A of the mycNgn3 determines virtually only nuclear protein localization. Therefore we can conclude that leucine 135 is the main amino acid for the binding of Ngn3 to exportin and that Ngn3 translocation from the cell nucleus to the cytoplasm in neurons is mediated by the exportin CRM1. This is in concordance with previous data showing that Ngn3 is retained in the cytoplasm of multiple endocrine neoplasia type 1 islet and pancreatic endocrine tumor cells and that tumor size is related with the level of CRM1 expression. It is of interest to note that L135A mutation had a stronger inhibitory effect on the nucleo-cytoplasmic transport of full length Ngn3 than on the nucleo-cytoplasmic transport of the NES2 fragment. The reason for this difference may be that Ngn3 has a nuclear localization site while NES2 does not. Therefore, in the case of full-length protein the NLS promotes its translocation to the nucleus. This may also explain why in cells transfected with wild-type myc-Ngn3, the myc fluorescence in the cytoplasm was Nuclear Export Signal in Neurogenin3 relatively decreased in comparison to cells transfected with myctag alone. Functional Consequences of Ngn3 Nucleo-cytoplasmic Transport The finding of Ngn3 shuttling between the cytoplasm and nucleus raises the question of whether this nucleo-cytoplasmic transport has a biological function. Other authors reported a patient with two heterozygous mutations in Ngn3 and a novel subtype of permanent neonatal diabetes associated with severe malabsorptive diarrhea. One of these mutation results in the substitution of leucine for proline at position 135. They also showed that electroporation with wild-type Neurogenin 3 provokes the differentiation of delaminating and hormone expressing cells outside the normal Oleandrin price pancreas, whereas electroporation with Ngn3L135P had no detectable effect on the chicken endoderm. We have found that L135A mutation, which reduces the nucelo-cytoplasmic transport of Ngn3, causes a decrease in the number of the primary dendrites and in 19276073 the number of synaptic inputs. This finding suggests that nucleo-cytoplasmic translocation of Ngn3 during neuronal development is involved in the initiation and growth of dendrites. Ngn3 and the Cytoskeleton The finding that the inhibition of nucleo-cytoplasmic transport of Ngn3 reduces dendritic development leads us to analyze whether Ngn3 may interact with components of the cytoskeleton. Immunocytochemical analyses showed that Ngn3 colocalizes with tubulin in cultured hippocampal neurons. The colocalization was detected in the perikaryon and the neuronal processes, including the growth cones. On the contrary, we did not detect colocalization of Ngn3 with actin filaments. In addition, Ngn3 increased in parallel with b-tubulin in the insoluble fraction of cell lysates from neuronal cultures or brain homogenates that were treated with paclitaxel to induce tubulin polymerization. These findings suggest that Ngn3 is associated with microtubules. Canonical microtubule binding motifs are present in MAP and tau proteins, but we have been unable to find such motifs in Ngn3. However, other proteins that lack canonical microtubule binding motifs, such 24726384 as kinesins and myosins, are also able to associate to microtubules. Furthermore, the interaction of Ngn3 with microtubules can be indirect and mediated by i