Assessment of Membrane Fluidity in Individual Yeast Cells by Laurdan Generalised Polarisation and Multi-photon Scanning
Here we describe techniques that we developed for monitoring membrane fluidity of individual yeast cells during environmental adaptation and physiological changes. Multi-photon scanning fluorescence microscopy using laurdan as a membrane probe enables det
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Assessment of Membrane Fluidity in Individual Yeast Cells by Laurdan Generalised Polarisation and Multi-photon Scanning Fluorescence Microscopy R. P.
LEARMONTH AND E. GRATTON
Here we describe techniques that we developed for monitoring membrane fluidity of individual yeast cells during environmental adaptation and physiological changes. Multi-photon scanning fluorescence microscopy using laurdan as a membrane probe enables determination whether fluidity changes seen by spectroscopy reflect universal responses or changes only of subpopulations. Yeast membranes are a primary site of environmental response and adaptation. Using fluorescence spectroscopy with DPH polarization and laurdan Generalized Polarization (GP), we previously found rapid "average" membrane fluidity modulation in yeast populations during growth and in response to nutrients or environmental stresses. To determine whether such responses reflect all cells we conducted the first multi-photon scanning fluorescence microscopy study of yeasts, measuring laurdan GP. We assessed membrane fluidity responses of individual yeasts related to growth phase, heat stress and ethanol stress. Average fluidity decreased as cultures aged, however the decreased fluidity was due in some cases to an increasing proportion of uniformly low fluidity (high GP) cells, which were shown by vital dye to be dead. When yeasts were heat stressed, the mean laurdan GP increased in all cells, thus the entire population evidenced damage (viz. decreased membrane fluidity) to the same degree. On the other hand, with ethanol stress fluidity increased (GP decreased) on exposure of cells. All cells were affected although not to the same degree, and with variable recovery. The recovery assessed from GP microscopy was highly variable, and greater by that seen by spectroscopy.
R. Kraayenhof et al. (eds.), Fluorescence Spectroscopy, Imaging and Probes © Springer-Verlag Berlin Heidelberg 2002
242 R. P. Learmonth and E. Gratton
14.1
Introduction
In order to provide background to these studies, we need to introduce three major concepts: the rationale for studying yeast membranes and their fluidity; the use of multi-photon scanning fluorescence microscopy; and the analysis of laurdan Generalized Polarization (GP). In brief, using fluorescence spectroscopy we previously studied membrane fluidity modulation in baker's yeast, brewer's yeast, and in Saccharomyces cerevisiae S288c for which the genome sequence is known. We determined membrane fluidity by fluorimetry, measuring polarization of DPH (l,6-diphenyl-l,3,5-hexatriene) fluorescence. More recently, to reduce problems with cell density-dependent scattering of the polarized light, we utilized the environmentally sensitive spectra of laurdan (6-lauroyl-2-dimethylamino naphthalene). We found rapid membrane fluidity modulation in yeasts in response to environmental stresses (heat and ethanol), during growth in batch culture and as a physiological response to glucose availability.
14.1.1 Yeast Membrane Fluidity
The plasma membrane provides t
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