TP C4: Microdomain calcium signalling in a secretory protozoan cell: Identification and functional localization of membrane proteins in cortical calcium stores

Description

The co-assembly of cortical calcium stores and of immediately releasable dense core secretory vesicles on the cell membrane is a striking feature of Paramecium cells and prerequisite of their extremely synchronous (80 ms) Ca2+-dependent exocytosis upon stimulation. We established rapid depletion of Ca2+ from subplasmalemmal stores, superimposed by "store-operated Ca2+-influx". The Paramecium genome project allowed us to clone part of two putative Ca2+-release channels with partially unusual characteristics. We plan to isolate the full length clones and their characterization, together with antibody production for localization and functional studies, functional knock-out by gene silencing and over-expression as GFP-fusion proteins. The hypothesis under debate that F-actin may link calcium stores to the cell membrane will be tested by overexpression and knock-out of cloned actin genes. This will be combined with analysis of Ca2+-dynamics upon stimulation, using fast confocal fluorochrome analysis and EM-based energy-dispersive x-ray microanalysis. We expect to provide first evidence of a site-directed Ca2+-release in a secretory system, which depends on the regular microzonal arrangement of the structural components involved.

Institutions
  • Department of Biology
Funding sources
Name Finanzierungstyp Kategorie Project no.
SFB third-party funds research funding program 582/03
Further information
Period: 01.07.2003 – 06.03.2011