Topic chosen: Proteins that are involved in the formation of tricellular junction in developing Drosophilia melanogaster’s wing imaginal disc.
We know:
Gliotactin is positioned onto bark-beetle at the tri-cellular junction during development. However Gliotactin does not directly interact with bark-beetle. The intracellular domain of Gliotactin contains a PDZ binding motif domain which interacts PDZ-containing proteins, such as scribbled and discs-large.
SPECIFIC QUESTION:
Using blast to find drosophila proteins that are similar to those that are known to interact with human homolog of discs-large, potential drosophila proteins that interact with drosophila disc large is identified. This project attempts to identify which proteins are potentially interacting with gliotactin during the formation of the tricellular junction. A falsifiable model will be proposed.
HOW IS THIS QUESTION NOVEL AND ORIGINAL?
The question of how gliotactin is position onto barkbeetle at the tricellular cellular junction during development is a known mystery. While other researchers in Auld lab have attempted to understand which proteins may be interacting with the tricellular junction, a interspecies blast has yet been performed. This approach to fish for the proteins that are important to the formation of tri-cellular junction is novel.
POTENTIAL IMPACT OF THE PROPOSED QUESTION (WERE IT TO BE ANSWERED BY YOUR PROPOSED EXPERIMENT):
Although vertebrates do not have tri-cellular junction, they possess tight junctions which are analogous to the tri-cellular junction. In both system, fluid permeability control is affected when junctions fail to form. Some possible consequences include exposure to toxic metabolites, loss of control over ion exchange and homeostasis. These results to lethality and disease. In understanding which proteins interact with gliotactin, we can understand how the tri-cellular junction is formed during development.
Furthermore, human discs-large have been extensively studied as it is a known tumour suppressor. In Drosophila, it has been shown by Woods and colleagues (2006) that Drosophila Dlg mutations causes overgrowth in the developing larvae brain and imaginal discs. When dlg-mutant imaginal discs were transplanted into wildtype flies, cancerous outgrowths that mimic metastatic tumours were observed by Woodhouse et al. (1998).
While Dlg is not the master gene to tri-cellular junction formation, it is beneficial to understand the induction hierarchy that may control its function. The identification of proteins that may inhibit or enhance the function of discs large may be helpful in cancer research.
HYPOTHESIS:
Veli proteins, RE30311p, RE51991p, X11L proteins, Jagar, alpha-actinin and moesin proteins are necessary in the proper positioning of Gliotactin at the tri-cellular junction.
EVIDENCE ON WHICH THE HYPOTHESIS IS BASED (INCLUDE REFERENCES):
1) The alignments from blasts (refer to supplemental word document)
From this point onward, my research is based on doctoral dissertation “Tricellular Junction Regulation, Signaling and Scaffolding” by Dr. Zohreh Sharif Khodaei.
In Zohreh’s dissertation, she proposed that Dls and scribbled are involved in the tricellular junction for the bark-beetle and Gliotactin to be properly places. I will do the proper citation later.
2) Veli proteins, RE30311p, RE51991p, X11L proteins contain PDZ domains which likely interacts with the PDZ binding motif of Gli since it has also been suggested that Gliotactin interacts with Dlg and Scrib PDZ domains at the SJ strands through its C-terminal PDZ binding motif (p.15) This hypothesis is suggested because PSD-95 (a homologue of Dlg) is a binding partner of Neuroligin (Nlg; a homologue of Gliotactin) by PDZ binding interactions (p. 16)
3) when PDZ binding motif of Gliotactin is defective, extensive endocytosis occurs to form large intracellular vesicle. It has been suggested that interaction between Gliotactin and Dlg stabilizes Gliotactin (p.86)
4) there are 4 PDZ domain in Scribbled (Scrib) that mediates protein interaction and are necessary to target Scrib to septate/bi-cellular junction (p.10) perhaps PDZ domain conservation is related to targeting proteins to junctions.
PREDICTION(S):
Because veli proteins, RE30311p, RE51991p have highly conserved domains, they likely interact with Gliotactin’s PDZ binding motif. Jagar contains SH3 domain which is likely to interact with proline rich motif. Alpha-actinin and moesin had high percent identity and alignment score conservation and interacts with the cytoskeleton. In binding the model, these probably indirectly interact with Gliotactin but help pull it into place by interacting with microtubules or actin filaments.
EXPERIMENTAL APPROACH TO TEST PREDICTION (INCLUDE ANY DETAILS THAT YOU HAVE WORKED OUT SO FAR):
First, create a stock fly line that has engrailed-GAL4/balancer. The stock genotype can be used to cross with UAS-geneRNAi. This knocks out the candidate gene’s mRNA at the posterior compartment. The anterior compartment acts as an internal control. The wing imaginal discs can be dissected from 3rd instar larvae and immunostained with antibodies specific to Gliotactin and Disc-large. The knockdown phenotype can be observed. After identifying which proteins delocalizes Gliotactin and Discs-large at the wing imaginal discs. To identify if each protein directly interacts with Gliotactin and/or disclarge, PLA can be performed with antibodies specific to proteins that has passed the screen and antibodies targeting Gliotactin and Discs-large.
Need a technique to track microtubules and gliotactin in vivo
LIST OF RELEVANT PRIMARY AND REVIEW ARTICLES READ, AND SUMMARY OF RELEVANT INFORMATION FROM EACH (this is the start of the annotated bibliography that you will need to include in your portfolio):
I’m organizing this part.
POTENTIAL WAYS TO MAKE YOUR QUESTION KNOWN TO THE PUBLIC AT LARGE (e.g. TO YOUR NON-BIOLOGIST FAMILY AND FRIENDS):
I will start a research blog to conclude my findings
I can also write a review article and post it on my facebook
ANY OTHER PARTS OF THE PROJECT COMPLETED SO FAR:
Supplemental documents, and all the blasts and alignments
ANYTHING YOU WOULD LIKE SPECIFIC FEEDBACK ON:
Experimental design