A study of 40LoVe's role in Xenopus development and the development of a novel intein based method for the in vivo conjugation of Quantum Dots to target proteins

Samba is a Xenopus hnRNP that was recently identified and has been shown to inhibit animal cap spreading and affect neural crest migration. It is expressed in all developmental stages and the expression appears elevated during neurula stages. At early neurula stages Samba is concentrated at the neural plate and later in the neural and neural crest tissues. At tail bud stages it is restricted in neural and neural crest derivatives including the brain, the spinal cord, the eyes and the brachial arches. Here we examined the protein localization at the cellular and embryonic level and also explored the role of the protein during development. We show that the protein is localized in the nucleus, the cytoplasm and the plasma membrane. It becomes associated with microtubules during mitosis and it is transported in axons. We also show using FRAP and FLIP that Samba shuttles between the cytosol and the nucleus consistent with a role as an hnRNP. Loss of function experiments using antisense Morpholinos leads to defective neural development in agreement with the elevated expression of Samba in neural tissues. However, we also show that the Morpholino down regulates the splice variant 40LoVe and a paralog, hnRNP AB, which shares 93% identity with 40Love. Despite the high homology between 40LoVe/Samba and hnRNP AB, the proteins localize in distinct manners suggesting distinct functions in the embryo. We map the differences between the two proteins to the c-terminus and show that the neural phenotype is a consequence of 40LoVe/Samba downregulation arriving at the surprising conclusion that 40LoVe/Samba and hnRNP AB despite their extremely high homology are in fact functionally distinct. The aforementioned project required the application of advanced imaging modalities including the use of fluorescent protein fusions for live imaging, FRAP and FLIP and highlighted the limitations of protein fluorophores with respect to photostability and brightness. Quantum Dots (QDs) are nanometer semiconductor nanocrystals with ideal optical properties for use in biological imaging. However no methodologies exist that will allow the covalent and site specific conjugation of QDs to target proteins in vivo. The second part of this thesis focused on developing a methodology that would resolve these issues. We describe an intein based method to site-specifically conjugate QDs to target proteins in vivo. This approach allows the covalent conjugation of any nanostructure and/or nanodevice to any protein and thus the targeting of such material to any intracellular compartment or signaling complex within the cells of the developing embryo. The C-terminus half (IC) of the intein was conjugated to QDs in vitro. IC-QD's and RNA encoding PH-IN were microinjected into Xenopus embryos. In vivo intein-splicing resulted in fully functional QD-PH conjugates that could be monitored in real time within live embryos. Use of Near Infra Red (NIR)-emitting QDs allowed monitoring of QD-conjugates within the embryo at depths where EGFP is undetectable demonstrating the advantages of QD's for this type of experiment. (author)