Elizabeth’s BioBlog

BioMinds Research Day

During the BioMinds Research Day, we had the opportunity to spend some time with leaders in the biotechnology industry, mentors from other universities and also students that had shared similar experiences. During the poster session I had the opportunity to visit and evaluate 2 posters from fellow students from other BioMinds campuses.

The first poster was presented by P. Silen Rivera and was titled: Prevalence of CYP2C19 Gene Polymorphisms in the Puerto Rican Newborn Population. The research was based on finding the prevalence of this gene, which affects the metabolism of certain drugs in the body, across several localizations in Puerto Rico. The samples in each town were collected randomly but representative of the town’s population. A broader and future goal of studies like this is to provide the base for personalized medicine where the person’s medical records can state that the abscence of certain genes can affect their ability to metebolize the prescribed medication. The poster had all of the necessary components and was well presented.

The second poster was presented by Beatriz Perez and was titled: “Soil Mite Fauna in Coastal Forests in Puerto Rico. The purpose of this study was to find the different species of soil mites and their localization in coastal forests. They discovered that the abundance and distribution of certain species in an alloted area varied over time. The presenter emphasized that soil mites differ greatly from so called “bed mites” and that they do not directly affect the human population. The poster had all of the necessary components and was also well presented.

I was not able to visit the third poster because it was not at the assigned spot and if it was moved to another spot, I wasn’t able to find it. The title was Development of a Nucleic Acid Biosensor for the Electrochemical Detection of Salmonella, presented by J. Ortiz.  

Overall the experience of presenting my poster and of reading about other student’s research was a great end to my time as a BioMinds student.

More Pictures

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

Reaching the finish line

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.

Abstract

        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. 

The Final Stretch (sort of)

It may be my last semester in the BioMinds program but it certainly is not the end of my project. Looking back on my past two semesters I realize that research is a lot more challenging and time-consuming that one might think as a novice. However, it is definitely worth the effort. Finally being able to see for myself the magnificent myopodia in action is just the beginning, but what a wonderful beginning for me! The semester has barely started and I already know that it will be full of great progress.. and more important.. RESULTS!!! This next few months I hope to be able to do in vivo  injections of various antibodies to cell adhesion molecules (CAMs) that are critical to neuromuscular synaptogenesis. As we identify and characterize the distribution of these CAMs we will be a step closer to understanding their individual roles in the process of creating the neuromuscular synapse. 

All the work and effort of past semesters are the base on which we can now work on. Our final objectives could’ve never been reached without setting up the microscopes, taking care of our Drosophila population, installing gas lines, and of course all the hours of practice of managing tiny little embryos and fussy little larvae. 

This semester I will be fine tuning my skills in needle pulling and beveling, in vivo embryo injections, and of course (my favorite) fluorescence confocal imaging. All of these techniques require lots of practice and dexterity because of all of the variables involved in each step. It is interesting and somewhat fun the hours of trial and error one goes through before getting it right, but after you are able to master it, everything else will just fall into place. 

Nature’s Wonders

After a semester of overcoming obstacles and setbacks I have learned that scientific research is unpredictable. During the past months, I have been reading scientific literature, taking care of our Drosophila cultures, searching for necessary antibodies, and immunostaining mutant embryos. While revising articles I found information on antibodies aganinst cell adhesion molecules such as Fas II, Fas III, N-cadherin, connectin, capricious, DSCAM. These cell adhesions molecules are important factors in neuromuscular synaptogensis because they mediate processes such as axon path-finding and synaptic targeting. We have found that these antibodies are necessary for our project because by localizing them and describing their behavior during the synapse formation we might have a better understanding of their individual roles in neuromuscular synaptogenesis.

The immunostaining of Drosophila embryos with anti-myosin solution gave unexpected results. Although we were unable to distinguish the presensce of myosin in the muscles we found that the embryos’ tracheas were clearly immunostained. The trachea, which was still in formation, showed filopodial extensions in search for the adjacent cell that will close the tubular structure. Here I include some images of the immunostained Drosophila trachea that include the filopodial extensions. You might notice that in these pictures, the trachea looks like a swan inside the embryo

 

Anti-myosin staining, M12 embryo, trachea

Snapshot, trachea 3DEmbryo trachea 3D

Regarding our laboratory, we are now completely set up and stocked and ready to begin a period of continous experimentation. The Drosophila population is mite-free and thriving. We still have some problems with fungal contamination in some vials but we are mantaining that under control.