1. Lab Meeting Michele Rodney 4/14/15

2. Projects I. Receptor Synaptic GRASP UAS-CG4356-4HA-15GS-sp11 flies (muscarinic Acetylcholine Receptor, A-type) II. Suntag Long linker for CG3822-3xspll (DkaiRD) III. Identify Novel Kainate Receptor Auxiliary Proteins – Tm5c Single expression profile

3. GRASP V3- receptor GRASP CG4356-muscarinic acetylcholine receptor design ~5.3Å 47Å ~124.8Å HA tags Presynapti c membrane -Tm mACh R ~200Å 45Å ? Postsynaptic membrane – T5 15X GS Linker Split GFP 1-10 Split GFP 11 Synaptobr evin

4. UAS-CG4356-4HA-15GS-sp11 flies (muscarinic Acetylcholine Receptor, A-type) Have approx. 22 independent lines Have lines with transgenes on X, 2 nd, and 3 rd chromosomes Set up crosses to balance and make true- breeding stocks

5. yw UAS-mCD8GFP T5-G4 (42H07) attp2 ----------------- ; ------------------------------------; ------------------------------- yw CyO Tm yw C1-3 LV, lexAop-syt-sp1-10 T5-G4 (42H07) attp2 ----------------- ; ------------------------------------; ------------------------------ yw CyO Tm Surface localization Activity GRASP L2->Tm2-> T5 L3->Tm9-> T5 To Test: CG4356-4HA-15GS-sp11 surface localization GRASP activity

6. SunTag Lets us do live imaging of receptors/proteins of interest HA tags mAChR Split GFP 1-10 cytosoli c Split GFP 11

7. HA tags mAChR Full length GFP Fluoresces

8. Longer Linker for DkaiRD-3Xsp11 GS HA GGSGG sp11 GGSGG 3.8A 19.8 8 45 8 sp1-10: 48.6 GS HA GGSGG sp11 GGSGGSGGSGGSGG 3.8 19.8 8 45 ~6.9*4+GG=27.6+GG DkaiRD CG3822 DkaiRD CG3822 -Sp11-GGSGG-sp11-stop ------ Ting

9. III. Goal: Identify Novel Kainate Receptor Auxiliary Proteins Auxiliary Proteins are are characterized by the following criteria: 1. Do not serve as an integral component of the transduction pathway 2. Remains stably associated with the receptor it regulates 3. Affects multiple aspects of receptor pharmacology, function and subcellular trafficking or targeting 4. Co-assembly with receptor is necessary for proper receptor functioning Copits&Swanson(2012) Screened existing literature for vertebrate proteins implicated as glutamate receptor auxiliary proteins Selected fly homologs Looked at expression levels in adult brain and L3 larvae CNS

10. Mammalian ProteinFunctionFly Homolog Fly Atlas Microarray: Expression in Adult head RNAseq: Expression in L3 Larvae CNS TARP Gamma 8; Stargazin Regulates trafficking and gating of AMPA receptors Stg1 (CG33670)LowVery Low CNIH2/CNIH3T1 TARPCNI (CG5855)Moderate GSG1LAMPAR Auxiliary SubunitStim (CG9126)Moderate SNAP25 Synaptosomal associated protein that regulates neurotransmitter release SNAP24( CG9474)N/AModerately High KRIP6 Interacts with GluK2 subunit to regulate kainate receptor function Dbo (CG6224), High, Moderate Moderate, KLHL18 (CG3571)Low PSD95 Scaffolding protein at synapse Dlg1 (CG1725)ModerateModerately High regulation of synaptic growth at neuromuscular junction (Bachmann et al., 2010) Dlin-7 (CG7662)HighModerate regulation of synaptic growth at neuromuscular junction (Bachmann et al., 2010) Metro (CG30021)HighModerate PICK1 aids in AMPAR clustering (Xia et al. 1999)PICK1 ortholog (CG6167) Low GRIP1 "AMPA receptor binding proteins potentially involved in the targeting of AMPA receptors to synapses" (Dong et al., 1999) GRIP, Glutamate receptor binding protein (CG14447)Low

11. Tm5c single cell expression profile for candidate genes As Tm5C expresses kainate glutamate receptors, it is a good system to identify potential kainate auxiliary proteins First need to see if the protein is expressed in Tm5c

12. Primer Design to check expression of candidate genes in Tm5c Need to be highly specific and produce a single band Need to span at least 2 exons so gDNA product can be distinguished from cDNA product Can be used with Taqman test External primers produce product of 400-600bp Nested primers produce product of 100-300bp

13. Testing of Primers on CS whole head cDNA first (PM) 1 2

14. 3

15. Gene External Primers ( from cDNA) External Primers (from gDNA) Nested primers ( from cDNA) Nested Primers ( from gDNA) Stargazin405474201270 CNI34753968137 Stim486553169236 Snap 24450 164 Dbo463601171309 2 500 bp

16. Tm5c single cell expression profile for candidate genes Make cDNA from single Tm5c cell selected by PM Check quality of sample – PCR with mCD8-GFP, Rp49, vGlut, Cha, Repo primers Test candidate gene specific primers on Tm5c single cell cDNA Once we know the qualitative expression profile, we can determine quantitative with Taqman PCR

17. Checking Tm5c cells picked by PM on Friday 500 bp mCD8-GFP 2 nd round Rp49 2 nd round mCD8RP49 cDNA266253 gDNA266315 Moyi & Michele

18. 500 bp ChAT 2 nd round nestChAT cDNA272 gDNA336 Moyi & Michele

19. vGlut 2 nd roundRepo 2 nd round nestvGlutRepo cDNA339552 gDNA807552 500 bp Moyi & Michele