Identification and Analysis of Dnaj-2 (cg10565) as a Regulator of Synapse Development in the Drosophila Larval Neuromuscular Junction

Open Access
Author:
Lee, Joyce
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Scott Brian Selleck, Thesis Supervisor
  • Ming Tien, Honors Advisor
Keywords:
  • Drosophila
  • Neuroscience
  • Genetics
Abstract:
The Drosophila larval neuromuscular junction (NMJ) is a powerful model system for the study of synaptic development generally and the assembly of post-synaptic membrane specialization specifically. Earlier work demonstrated that decreased expression of Akt1 in muscle tissue causes specific mislocalization of one particular glutamate receptor Subunit, GluRIIA.1 To search for additional signaling components affecting GluRIIA delivery and assembly into a functional glutamate receptor, an RNA interference (RNAi) genetic screen of ~500 genes was performed. The organization of the post-synaptic membranes of the test animals was assayed using fluorescence confocal microscopy and immune-detection of GluRIIA. From the screen, a previously uncharacterized gene, DnaJ-2 (CG10565), was identified as a potential regulator of GluRIIA localization at the NMJ. This gene encodes a protein belonging to the J-protein chaperone family. In order to further study DnaJ-2, we have generated mutants via imprecise P-element excision. The putative mutants were screened by PCR genotyping. After the initial round of mutagenesis crosses, the genetic cross scheme and assaying methods were optimized in order to increase the frequency of the imprecise P-element excision detection. At present we have identified 4 excision mutants that disrupt the DnaJ-2 sequence, ranging from a 67 bp deletion to a 1.8kb deletion. Additionally, constructs containing specific mutations of the DnaJ-2 sequence were generated and placed into transgenic flies. These lines as well as the mutant will be useful for future study of DnaJ-2.