1. Research Advisor: Dr. Charles Singleton
Basic Science: An Investigation Into Ammonium Transporter Expression and Function During Development in Dictyostelium discoideum
Colin Dinsmore
BSCI 296
Research Advisor: Dr. Charles Singleton
April 27, 2007

2. Dictyostelium Life Cycle
Copyright, M.J. Grimson & R.L. Blanton

3. Cell Differentiation and Sorting

4. Ammonium Transporters
AmtA- develops quickly, rarely forms slugs, forms small structures, and is insensitive to high ammonia
AmtC- development arrests at slug stage
AmtA-/AmtC- has 1-2h transitional period, followed by culmination, and is insensitive to high ammonia
RdeA
AmtA
( NH3)‏
DhkC
RegA
AmtC
( NH3)‏
cAMP
AmtB
????
PKA
nSTATa
CudA
Culmination
adapted from Kirsten et al., 2005

5. amtC Expression Pattern
Follstaedt, et al.; Kirsten, et al.

6. amtC/AmtC/YFP Pattern
Mound Slug Second Finger
Ax4
Ax4/C-40
Ax4/C41

7. Conditions Favoring Migration as Slug
Ax4/C40 - Slug Stage
Development in the dark
Development on 1.5% water agar

8. Pattern in AmtC- Background
Standard Conditions, 14h
Standard Conditions, 18h
Dark, 14h
Water Agar, 13h

9. Promoter Analysis Conclusions
The amtC promoter is subject to regulation during the transitional period by a factor that is itself regulated by the developmental stage of the organism, not presence of mature AmtC protein
This factor may or may not regulate other genes – further studies are needed
Loss of prespore expression perhaps due to independent, mature AmtC regulated mechanism

10. Mechanism of action: The search for binding targets
Purely import/export activity or is there a signaling component?
If signaling component, direct or indirect action on DhkC?
Use fusion proteins to detect potential binding targets for Amts
RdeA
AmtA
( NH3)‏
DhkC
RegA
AmtC
( NH3)‏
cAMP
AmtB
????
PKA
nSTATa
CudA
Culmination
adapted from Kirsten et al., 2005

11. Mechanism of action: The search for binding targets
No His-tag
His-AmtC
GST-fusion pulldowns suffered from high background
Expressing these proteins in vivo may alleviate this problem
His-tagged versions may be a more viable alternative (His-AmtC shown)‏
TINE
1M NaCl 50 °C
TINE Elutions [imiadazole]
1M NaCl 50 °C

12. Future Work Further amt promoter analysis
LacZ studies
Development in other backgrounds
Transcription factor pulldown and subsequent ChIP (on chip) experiments
New affinity chromatography approaches
Repeat His-AmtC experiment, if positive ID protein(s) by mass spectrography and make other His-Amt constructs
Pulldown with TAP-Tag labeled DhkC using available plasmids

13. Acknowledgments Dr. Charles Singleton Jan Kirsten Yanhua Xiong
Fellow Undergraduates