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Chemical Genomics Approach to Endocytic Pathways in Arabidopsis thaliana using
Nicotiana tabacum pollen
R.J. Washington, M. Brown, N.V. Raikhel
NSF-REU Program 2008
Source: News in Science, 8 January 2002www.abc.net.au/science/news/stories/s453641.htm
ERGolgi
vacuolePVC
TGN
EndosomesPlasma
membrane
PIN2
RIP1
AUX1BRI1
A closer look at the endomembrane system
EXPERIMENTAL APPROACHClassical genetics vs Chemical Genomics
Functional protein
Genetic mutation Chemical interaction
Non-functional protein
Classical genetics Chemical Approach
Cl
Cl
Pollen tip polarized activity
Germination: Three tier activity
– Zone 1- Apical: New materials deposited at apical end via secretory vesicles
– Zone 2- Sub-apical: actin filaments, mitochondria and EM organelles
– Zone 3- Shank: composed of actin filaments
All zones constantly participate in concert to propel tube growth via the dynamic endocytic/exocytotic processes
Model from Cole and Fowler (2006) Current Opinion Plant Biol. 9:1-10
Primary Chemical Screen
WT Tobacco Pollen
BD Pathway microscope
Chemical Library
Inhibition of pollen germination
384 well plate
Aberrant pollen morphology
+
Secondary Chemical Screen
RIP1:GFP marker Tobacco Pollen
Mislocalization of marker
96 well plate
Aberrant pollen morphology
Examine with BD Pathway microscope
Primary Screen Inhibitors serially diluted
A B
+
Library Number of Compounds Primary Hits Primary
Percentage Secondary HitsSecondary Percentage
Diverset 20,000 218 1.09% 22/176 12.5%
Novacore 10,000 18 0.18% 0/18 0%
TimTec 10,000 33 0.33% 0/30 0%
LATCA 3650 89 [76] 2.4% 16/73 21.9%
CLICK 2768 15 0.54% 0/7 0%
TOTALS 46418 360 .78% 38/302 12.6%
Summary of library progress of pollen screens
ANTIMICROBIAL
3
ANTIFUNGAL
1
ANTICARCINOGEN
3
ANTIPARASITICAL
4
ANTIBACTERIAL
3
CYTOSKELETAL EFFECTS
2
TRANSPORT PROTEINS
4
OTHER EFFECTS
5
Bioactivity of secondary hit compounds from database screen inquiry
Based on structure database searchScreening database used in inquiry: Science Finder Scholar Program
Future Work
Chemical Screening of Arabidopsis seedlings− Root and vegetative tissues phenotype effects categorized for
each test compound− Marker lines imaged via confocal microscopy and analyzed to
determine localization effects
Plasma membrane TGN Bri-overexpression PIN2
Acknowledgements• Principal Investigator: Dr. Natasha V. Raikhel
• Mentor: Michelle Brown
• Lab members:Glenn Hicks, Marci Surpin, Mien van de Ven, Stephanie RobertJulie Sohn, Georgia Drakakaki, April Agee, Abel Rosado Rey
• Support Staff and Facilities: Dr. David Carter (CEPCEB Academic Coordinator, Microscopy/Imaging) Dr. Thomas Girke (CEPCEB Academic Coordinator, Bioinformatics) Institute for Integrative Genome Biology (IIGB)-Chemical Libraries Plant Transformation Research Center
• Support Funding: National Science Foundation