BBSRC Portfolio Analyser
Award details
Investigating the PrpS-PrsS (pollen and pistil SI determinant) interaction
Reference
BBS/E/J/000CA429
Principal Investigator / Supervisor
Professor Dale Sanders
Co-Investigators /
Co-Supervisors
Institution
John Innes Centre
Department
John Innes Centre Department
Funding type
Research
Value (£)
2,302
Status
Completed
Type
Institute Project
Start date
01/08/2010
End date
31/07/2013
Duration
36 months
Abstract
This project will examine the nature of PrpS and its proposed "receptor-ligand" type interaction with PrsS, using a range of live-cell approaches. We will use a range of live-cell approaches to study the SI determinant-ligand interactions in transgenic Arabidopsis pollen protoplasts, transfected animal cells and poppy pollen tubes transiently expressing PrpS-GFP using a biolistic approach. We will also use Alexa-tagged PrsS for some experiments. This will provide a detailed determination of the nature, localization, distribution and dynamics of the receptor-ligand interaction. (A) aims to establish the nature of PrpS as an ion channel We will express PrpS in a heterologous cell system (Xenopus oocytes and /or HEK cells) to build on preliminary data to firmly establish that PrsS functions as an ion channel. Patch- clamping will allow us to identify PrsS-induced activation and kinetics, channel conductance and selectivity and pharmacological properties. (B) and (C) focus on studying PrpS dynamics and interactions with PrsS and identifying amino acids/domains involved in recognition/function. We will use live-cell imaging using fluorescence microscopy, including: Bimolecular fluorescence complementation (BiFC), Total internal reflection fluorescence microscopy (TIRF-M), FRAP and FRET to study PrpS-PrsS interactions in detail. Site-directed mutagenesis on the predicted extracellular 35 aa loop and also making PrpS chimeras by domain-swopping this region between allelic variants (e.g. replace S1 with S3) will establish which changes result in loss or altered function or S-specificity. These approaches will allow us to establish if PrpS forms a multimeric complex, establish the subcellular localization/activity of PrpS/PrsS, examine probe real-time PrpS-PrsS interaction dynamics in live cells, and establish the nature of the allele-specificity of the PrpS-PrsS interaction.
Summary
unavailable
Committee
Not funded via Committee
Research Topics
Plant Science, Structural Biology
Research Priority
X – Research Priority information not available
Research Initiative
X - not in an Initiative
Funding Scheme
X – not Funded via a specific Funding Scheme
I accept the
terms and conditions of use
(opens in new window)
export PDF file
back to list
new search