Activity-dependent transcriptional dynamics in mouse primary cortical and hum...
Wsp Role in CNS Development
1. The Effect of the Wiskott-Aldrich Syndrome Protein
(Wsp) on the Developing CNS in Drosophila
Omar Johnson
Summer Independent Study Project
Bashaw Lab
2. Axon guidance at the Drosophila
ventral midline
• Axons must navigate correctly to
form a functional circuit.
• The ventral midline = an
intermediate choice point.
Corey
Goodman
4. Netrin-Frazzled signaling
• Netrin is secreted by
midline glial cells,
and attracts axons
expressing the
Frazzled receptor to
the midline.
lGGarbe et al. Development 2007 2007
5. Yeast Two Hybrid screen to identify
proteins that interact with the Fra ICD
DNA-
binding
Proteins
(9)
Nuclear
Proteins
(4)
Metabolic
/
Other
Proteins
(45)
Cytoskele
tal-
interactin
g Proteins
(9)
Ribosoma
l
Proteins
(20)
RNA-
binding
Proteins
(3)
• Colonies grow only if the library protein interacts
with the Fra ICD.
Chro
Gro
Med30
B52
FAK
Mars
WASp
6. Structure of the Drosophila Wasp
Homolog
Tal et. Al , Dev. Bio 243, 260-271 (2002)
• Structure of Wasp is characterized by a conserved domain arrangement.
• C-terminus contains a Wasp Homology domain for binding monomeric
actin followed by a short central domain (C) and an acidic (A) domain
which interacts with the Arp 2/3 complex
• N-terminus contains domains thought to be regulatory in nature and auto-
inhibits Wasp function by occluding the C-terminus.
7. Functions of the Drosophila Wasp
Homolog
• Myoblast fusion
• Axonal fasciculation and axonogenesis (through an
interaction with SCAR)
• Clathrin-mediated endocytosis
• Sensory organ development
• Regulation of synaptic growth at neuromuscular
junction
• Actin polymerization
8. Role of Wasp in Actin
Polymerization
Goley and Weich, Nature Reviews Mol. Cell Bio 7, 713-726 (Oct
2006)
9. Wasp Expression during
Embryogenesis
• Mesa et al. (2012) used anti-Wasp P5E1 antibody and confocal
microscopy to visualize Wasp expression in the Drosophila
embryo.
-Found Wasp was expressed
Uniformly in the cytoplasm.
-Enriched in the stomach,
Posterior midgut, and
Malpighian tubules in
Late stage embryos.
-No observed accumulation in
Neural or muscle precursors despite
known role in neurogenesis and
Myoblast fusion.
Mesa et al, Dev Dynamics 241:608-626, 2012
10. Wasp Expression in Drosophila
Embryos
• UAS-Wasp x apTMG
• Apterous neurons observed crossing the midline.
• Staining Protocol:
– Primary: GFP, Wasp P5E1, Wasp P3B1
– Secondary: Alexa 488, Cy-3 (Gt anti MS)
13. Wasp Expression in Drosophila
Embryos
• e1Wasp/TM3Ubx x e1Wasp/TM3Ubx
• No significant defects observed.
• Staining Protocol:
– Primary: Fas II and HRP
– Secondary: Alexa 488 (Gt anti MS)
14. Wasp Expression in Drosophila
Embryos
• UAS-Wasp X ScaGal4
• No significant defects observed
• Staining protocol:
– Primary: Wasp (P5E1)
– Secondary: Alexa 488 (Goat anti MS), Cy -3 (Anti HRP)
15. Cell Transfection
• S2R+ cells were transfected with Wasp, Fra-Myc, and Fra-ICD
plasmids to show expression and localization of proteins.
• Protein expression was induced using CuSO4
• Results show that these proteins were expressed throughout
the cell.
Wasp Fra-Myc Fra-ICD
16. Co-transfection of S2R+ cells
• S2R+ cells were co-transfected with Wasp and
Fra-ICD plasmids to show co-localization and
possible protein-protein interactions.
Cells expressing both
proteins
Cells expressing Fra-ICDCells expressing Wasp
17. Immediate Next Steps
• Wasp/Fra double mutants embryos
• Recombinant Wasp embryos with egGal4
• Score ectopic crossing in UAS-Wasp x apTMG
embryos
• Co-IP assay to show protein-protein
interaction between full length Fra and Wasp
18. Acknowledgements
• Greg J. Bashaw, PhD
• Samantha Russell
• Madhavi Gorla, Ph.D.
• Elise Abeille, Ph.D.
• Melissa Hernandez
• Celine Santiago
• Karina Chaudhari
• Kevin Wiles
• Yixin Zang
• Katherine Blocklove
• Nick O’ Neill
• Ethan Rohrbach
• Kuan Yu
• Bob Herbstritt
Editor's Notes
Correct axon guidance is necessary for neurons to synapse with their targets. Bilateral motility, decision points, midline crossing, etc.
Yeast two hybrid screen to identify proteins that interact with the Fra ICD that has a point mutation making it dead for transcriptional activation and a cDNA library from embryonic drosophila. Identified about 100 proteins that I’ve separated into different functional classes. It’s clear that both proteins involved in canonical as well as non-canonical have been identified. FAK …mars… Nuclear proteins, and DNA-binding proteins.
Schematic for Drosophila Wasp homolog
COOH terminus acts as the effector domain
NH2 terminus acts as a regulatory domain by occluding the C-terminus
Binding of anionic phospholipids or the small GTPase Cdc42 frees the COOH terminus to interacts with monomeric actin via the Wasp Homology domain or the Arp2/3 complex via the short central C domain and the Acidic A domain.
Does overexpression of Wasp cause ectopic crossing of apterous neurons?
If so, is it a result of interactions between Wasp and Fra.
