Project Details
Projekt
Imaging the dynamics of actin-associated proteins in motile cells using ultra-fast, multi-spectral TIRF microscopy
Applicant
Professor Dr. Stefan Diez
Subject Area
Biophysics
Term
from 2007 to 2010
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 38396385
It is the goal of the project to study the recruitment and the dynamics of actin-associated proteins in the cortex of motile Dictyostelium Discoideum cells with high spatial and temporal resolution. In particular, we will focus our studies on proteins involved in actin dynamics with respect to the formation of leading edges, traveling waves, patches and networks. We aim to visualize the participating proteins, such as myosin-IB, with single molecule resolution and expect to gain insight into their possible organization into multimeric complexes as well as into their involvement in actin organization. Because preliminary experiments indicate the majority of actin patches being preceded by spotty structures containing Clathrin, the study of Clathrin dynamics will be a second major part of the project. Analysis of the motility of the Clathrin structures will enable us to distinguish various modes of movement in the vicinity of the plasma membrane and to study microtubule-dependent motor activities in intact cells. Simultaneous imaging of actin and Clathrin components using a dual-color fluorescence approach will allow for a better understanding of the involvement of dynamic actin structures in Clathrin-mediated endocytosis. To tackle these tasks, we will use highly-sensitive and ultra-fast fluorescence microscopy combining; (i) low background fluorescence imaging by widefiled total-internal reflection fluorescence (TIRF) illumination, (ii) ultrafast image acquisition of 100 frames per second and above by applying electron multiplied charge coupled device (EMCCD) technology, (iii) simultaneous multicolor imaging by spectral splitting and (iv) computerized algorithms for highly-parallel particle tracking with nanometer resolution.
DFG Programme
Priority Programmes
