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Optically invisible properties of membrane organization are revealed by TIRF‐FCS on sphingolipid‐interacting probes
Author(s) -
Kraut Rachel,
Manna Manoj,
Sankaran Jagadish,
Wohland Thorsten
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.585.11
Subject(s) - total internal reflection fluorescence microscope , membrane , fluorescence correlation spectroscopy , biophysics , diffusion , chemistry , c2 domain , biology , physics , biochemistry , molecule , organic chemistry , thermodynamics
The objective of this study was to observe sphingolipid behavior in cell membranes. For this, we generated the ganglioside‐binding probes SBD ( S phingolipid B inding D omain), derived from Alzheimer's Aß peptide, and TeNT46, from Tetanus toxin. Both these peptides display slow, bimodal diffusion behavior at membranes typical for ordered domain (l o ) probes, but show different lipid requirements for binding and slow diffusion. Fluorescence correlation spectroscopy (FCS) has sufficient time resolution to follow dynamics of these membrane domain‐associated probes, but it cannot discern features of lateral organization. We therefore measured a parameter called the ΔCCF (for difference in the cross‐correlation function), which detects anisotropic distributions of diffusion behaviors at the membrane, from correlations between neighboring pixels in a field obtained by TIRF‐FCS. ΔCCF shows that the high anisotropy of l o probes, like diffusion, is sensitive to domain disruption. Notably, heterogeneity revealed by ΔCCF responds differently from diffusion after various perturbations, and appears to detect changes to which traditional FCS is blind. Thus, two levels of organization at the plasma membrane are distinguishable by these methods: single point diffusion, vs. longer‐scale heterogeneity. Financial support provided by the Ministry of Education, and Nanyang Technological University, Singapore.