Elizabeth’s BioBlog

I have also put more images that have been recently acquired in the pictures page. You should go see them! 

After another month hard at work with every aspect of a drosophila laboratory we’ve progressed (on a scale from 1-5) a 3. We have put on hold the needle pulling and continued with embryo immunohistochemistry with different CAMs and myosin. I have acquired some images that show the presence of fas II (intracellular domain) in embryos at stages 13-15. In these images we can see that fas II is present at the central nervous systema and in all nerves throughout the body. The images acquired are shown below

Working at a laboratory with animal models has its complications and setbacks. Weekly, we confront many problems with the Drosophila population that need to be resolved immediately (or else we risk losing the stocks). We constantly have problems with factors like fungi in the bottles, mites in the stocks, food thats too hard or dry, flies dying for mysterious reasons, and to top that, problems with the fluorescence in specific lines of flies. Attending these issues takes time and effort that keeps me away from experiments. We have made new food, cleaned the bottles and tubes to get rid of the fungi and the mites, and isolated fluorescent larvae and embryos to make stocks that are expressing the GFP correctly. We hope that our continuos efforts in keeping our population have a positive outcome.

        The identification and characterization of key components in neuromuscular synaptogenesis has been the object of study in past decades. The field has been progressively advancing as processes such as axon path-finding and synaptic targeting are being better understood. However, researchers in post-synaptic dynamics and trafficking have yet much to discover, specifically in the study of protein transport to the post synaptic density (PSD). The PSD is a sub-cellular specialization at neuronal synapses thought to organize the postsynaptic signaling complexes required for accumulation of scaffold proteins such as PSD95/Dlg. During  the formation of the neuromuscular junction in Drosophila melanogaster, the PSD is assembled at the tip actin-filled filopodia called myopodia that are extended by the post-synaptic cell (muscle). Non-muscle myosin, a motor protein, mediates the intracellular transport of Dlg and may also be responsible for transporting various cell adhesion molecules (CAMs) throughout the myopodia and into the PSD. In this study we aim to understand the role motor proteins and CAMs play in muscle innervation. By identifying the specific myosin responsible for intra-myopodial trafficking and the spatiotemporal pattern for CAM transport, we can start to dissect the sequence of events that ultimately leads to a functional synapse. This new knowledge can lead to both clinical and industrial applications in different fields,such as bioengineering. To accomplish this we used fluorescent confocal imaging together with both in vitro and in vivo immunohistochemical staining using genetically encoded green fluorescent protein (GFP) bioprobes. This approach allows us to track and characterize the individual function of both myosin and CAMs in neuromuscular synaptogenesis. Also, by a developmental time course we can determine the stage and specific timepoint where the immunostaining becomes apparent.  Surprisingly, while looking for myosin in myopodia during a developmental time course we came upon myosin in extended filopodia of the developing foregut epithelium. A concentrated amount of myosin in the extended filopodia suggests an additional function in the closure of the tube-like structure. Additional studies are necessary to understand the role of myosin in this developmental context in Drosophila development. Furthermore, we are still in the process of identifying the particular myosin and CAMs involved in key processes in neuromuscular synaptogenesis. This project is sponsored by BioMinds.