Probing orientational behavior of MHC class I protein and lipid probes in cell membranes by fluorescence polarization-resolved imaging - PubMed (original) (raw)

Probing orientational behavior of MHC class I protein and lipid probes in cell membranes by fluorescence polarization-resolved imaging

Alla Kress et al. Biophys J. 2011.

Abstract

Steady-state polarization-resolved fluorescence imaging is used to analyze the molecular orientational order behavior of rigidly labeled major histocompatibility complex class I (MHC I) proteins and lipid probes in cell membranes of living cells. These fluorescent probes report the orientational properties of proteins and their surrounding lipid environment. We present a statistical study of the molecular orientational order, modeled as the width of the angular distribution of the molecules, for the proteins in the cell endomembrane and plasma membrane, as well as for the lipid probes in the plasma membrane. We apply this methodology on cells after treatments affecting the actin and microtubule networks. We find in particular opposite orientational order changes of proteins and lipid probes in the plasma membrane as a response to the cytoskeleton disruption. This suggests that MHC I orientational order is governed by its interaction with the cytoskeleton, whereas the plasma membrane lipid order is governed by the local cell membrane morphology.

Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

Schematic representation of (a) MHC_GFP_Rigid and (b) MHC_GFP_Flexible. (c) Typical polarimetry responses IX(α) and IY(α) measured for a varying incident polarization angle α, at a given point of a di-8-ANEPPQ-labeled cell membrane. (Continuous lines) Fits (see the Supporting Material). (d) Scheme of a membrane equator representing the molecular orientational cone-shaped distribution with the cone aperture angle ψ of fluorescent probes inserted into the membrane or attached to a membrane protein. The X and Y axes define the sample plane in which the excitation polarization is fixed. The angle ρ specifies the cone orientation in this frame. (e) Anisotropy ratio A(ψ) dependence for ρ = 0° (top line), ρ = 45° (middle line), and ρ = 90° (lower line). This function accounts for polarization distortions in the setup (see text).

Figure 2

Figure 2

Fluorescence image (top) and corresponding anisotropy images (bottom) of COS-7 cells labeled with (a) MHC_GFP_Rigid, (b) MHC_GFP_Flexible, (c) di-8-ANEPPQ without treatments and (d) MHC_GFP_Rigid, (e) MHC_GFP_Flexible, (f) di-8-ANEPPQ treated with latrunculin A. The typical fluorescence intensity varies between 200 and 500 K counts/s. Scale bar: 10 _μ_m.

Figure 3

Figure 3

Histograms showing the cone aperture angle distribution ψ before (plain histograms) and after the treatments (line histograms) with (a) hypotonic shock, (b) latrunculin A, (c) cytochalasin D, and (d) colcemid, for MHC_GFP_Rigid in both the endomembrane (left) and the plasma membrane (middle), and di-8-ANEPPQ in the plasma membrane (right). All measurements are performed at 37°C. In each case, the statistics are performed on 200 total areas taken within a population of 5–10 cells.

Figure 4

Figure 4

Proposed scheme for (a) MHC I and (c) di-8-ANEPPQ orientational behavior in the untreated case and (b) MHC I and (d) di-8-ANEPPQ after actin polymerization inhibition with Latrunculin A.

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