Assessment of Membrane Fluidity in Individual Yeast Cells by Laurdan Generalised Polarisation and Multi-photon Scanning Fluorescence Microscopy

Abstract

Here we describe techniques that we developed for monitoring membrane fluidity of individual yeast cells during environmental adaptation and physiological changes. Multiphoton scanning fluorescence microscopy using laurdan as a membrane probe enables determination whether fluidity changes seen by spectroscopy reflect universal responses or changes only of sub-populations.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.

Lipid Composition Analysis Reveals Mechanisms of Ethanol Tolerance in the Model YeastSaccharomyces cerevisiae

“…We next examined the effect of ethanol upon the fluidity of the yeast membranes as they grew in cultures with and without ethanol. We utilized the fluorescent dye, Laurdan, which has been used to study phase properties of membranes as it is sensitive to the polarity of the membrane environment (47). GP (Generalized Polarization) values, which correlate inversely with fluidity, were calculated at six timepoints during the growth of AJ4, MY3, MY14, MY26 and MY29 strains in GPY, GPY containing 6% ethanol and GPY containing 10% ethanol.…”

Genes mapping to boron tolerance QTL in barley identified by suppression subtractive hybridization

“…1998). Polyamines have also been implicated in changes in membrane fluidity affecting cell permeability and important activities such as nutrient transport and pH maintenance (Kakkar & Sawhey 2002; Learmonth & Gratton 2002; Wallace et al. 2003).…”

“…Spermidine and spermine are efficient antioxidants, which scavenge oxyradicals and protect membranes (Bors et al 1989;Ha et al 1998). Polyamines have also been implicated in changes in membrane fluidity affecting cell permeability and important activities such as nutrient transport and pH maintenance (Kakkar & Sawhey 2002;Learmonth & Gratton 2002;Wallace et al 2003). It is possible that these properties of polyamines could account for a reduction in passive uptake of boron and hence for the enhanced tolerance to boron toxicity.…”

Protein induced membrane phase transition facilitates leishmania infection

“…It has been established that an increase in MF results in the decrease in FA and GP. 10 Both assays (FA, Figure 1a and GP, Figure 1b) showed significant increase in MF in 12 h infected Mϕ (LD-M). Both assays also showed that the treatment of cholesterol partially offset the effect of LD infection (Figure 1a Interestingly, we found that KMP-11 mimicked the effect of LD.…”