4D RECONSTRUCTIONS OF ACTIN CYTOSKELETON IN LIVE YEAST CELLS

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Studies of actin cytoskeleton dynamics are the necessary to understand the function of the actin cytoskeleton in yeast. The results of these studies should be applicable not only to yeasts but to higher organisms due to homology of both systems. The specific aim of this project is to study the dynamics of the actin cytoskeleton in live yeast cells by means of 4-1) microscopy. Previously we were able to label the yeast actin patches with GFP and to demonstrate that they move. We also performed preliminary tracking of moving patches in 2-D and we were able to determine their speed (Waddle et al., 1996). 4-1) reconstructions should permit us to track the individual actin patches through multiple focal planes and for a long time. 4-1) tracking of patches should p6nnit us to distinguish between the disassembly of patches and movement out of the focal plane. 4-D tracking should also permit us to unambiguously document the instances of patch division, fusion, assembly and disassembly in live cell. We also want to study the impact of genotype and environment on the speed and character of patch movement. We would like to follow the individual patches labeled by photobleaching and photoactivation and establish the rate of protein turnover in the patches and a possible connection of actin assembly to patch movement. Our current computerized video-system does not permit the necessary speed of acquisition to perform the fast Z-scans necessary to track individual patches in 4-1). In our system, long-term observations at high magnification are limited by photobleaching of the GFP label and photodamage to the cells. Multi-photon laser-scanning fluorescence microscopy appears to be best suitable for a long-term observation without photobleaching and photodamaging. We hope that HAR high-quality video carneras will peffnit us to perfonn fast capturing of sequential focal planes. We intend to use IMR software for large-scale tracking of patches and 4-D reconstructions.