Lab Meeting

Michele Rodney4/14/15

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

GRASP V3- receptor GRASPCG4356-muscarinic acetylcholine receptor design

~5.3Å

47Å

~124.8Å

HA tags

Presynaptic

membrane-Tm

mAChR

~200Å

45Å ?

Postsynaptic membrane –

T5

15XGS Linker

Split GFP 1-10

Split GFP11

Synaptobrevin

UAS-CG4356-4HA-15GS-sp11 flies(muscarinic Acetylcholine Receptor, A-type)

• Have approx. 22 independent lines • Have lines with transgenes on X, 2nd, and 3rd

chromosomes• Set up crosses to balance and make true-

breeding stocks

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 GRASPL2->Tm2-> T5L3->Tm9-> T5

To Test:

• CG4356-4HA-15GS-sp11 surface localization• GRASP activity

SunTag• Lets us do live imaging of receptors/proteins

of interest HA

tags

mAChR

Split GFP 1-10

cytosolic

Split GFP11

HA

tags

mAChR

Full length GFPFluoresces

Longer Linker for DkaiRD-3Xsp11GS HA GGSGG sp11 GGSGG3.8A 19.8 8 45 8

sp1-10: 48.6

GS HA GGSGG sp11 GGSGGSGGSGGSGG3.8 19.8 8 45 ~6.9*4+GG=27.6+GG

DkaiRDCG3822

DkaiRDCG3822

-Sp11-GGSGG-sp11-stop

------

Ting

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 pathway2. Remains stably associated with the receptor it regulates3. Affects multiple aspects of receptor pharmacology, function and

subcellular trafficking or targeting4. Co-assembly with receptor is necessary for proper receptor

functioningCopits&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

Mammalian Protein Function Fly 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) Low Very Low

CNIH2/CNIH3 T1 TARP CNI (CG5855) Moderate Moderate

GSG1L AMPAR Auxiliary Subunit Stim (CG9126) Moderate Moderate

SNAP25Synaptosomal associated protein that regulates neurotransmitter release

SNAP24( CG9474) N/A Moderately High

KRIP6Interacts with GluK2 subunit to regulate kainate receptor function

Dbo (CG6224), High, Moderate Moderate,

KLHL18 (CG3571) Low

PSD95 Scaffolding protein at synapse Dlg1 (CG1725) Moderate Moderately High

regulation of synaptic growth at neuromuscular junction (Bachmann et al., 2010)

Dlin-7 (CG7662) High Moderate

regulation of synaptic growth at neuromuscular junction (Bachmann et al., 2010)

Metro (CG30021) High Moderate

PICK1aids in AMPAR clustering (Xia et al. 1999) PICK1 ortholog (CG6167)

Low 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 Low

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

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

Testing of Primers on CS whole head cDNA first (PM)

1 2

3

GeneExternal Primers( from cDNA)

External Primers (from gDNA)

Nested primers( from cDNA)

Nested Primers ( from gDNA)

Stargazin 405 474 201 270

CNI 347 539 68 137

Stim 486 553 169 236

Snap 24 450 450 164 164

Dbo 463 601 171 309

2

500 bp

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

Checking Tm5c cells picked by PM on Friday

500 bp

mCD8-GFP 2nd round Rp49 2nd round

mCD8 RP49

cDNA 266 253

gDNA 266 315 Moyi & Michele

500 bp

ChAT 2nd round

nest ChAT

cDNA 272

gDNA 336

Moyi & Michele

vGlut 2nd round Repo 2nd round

nest vGlut Repo

cDNA 339 552

gDNA 807 552

500 bp

Moyi & Michele