Local Mobility in Lipid Domains of Supported Bilayers Characterized by Atomic Force Microscopy and Fluorescence Correlation Spectroscopy

Abstract

Fluorescence correlation spectroscopy (FCS) is used to examine mobility of labeled probes at specific sites in supported bilayers consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid domains in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Those sites are mapped beforehand with simultaneous atomic force microscopy and submicron confocal fluorescence imaging, allowing characterization of probe partitioning between gel DPPC and disordered liquid DOPC domains with corresponding topography of domain structure. We thus examine the relative partitioning and mobility in gel and disordered liquid phases for headgroup- and tailgroup-labeled GM1 ganglioside probes and for headgroup- and tailgroup-labeled phospholipid probes. For the GM1 probes, large differences in mobility between fluid and gel domains are observed; whereas unexpected mobility is observed in submicron gel domains for the phospholipid probes. We attribute the latter to domain heterogeneities that could be induced by the probe. Furthermore, fits to the FCS data for the phospholipid probes in the DOPC fluid phase require two components (fast and slow). Although proximity to the glass substrate may be a factor, local distortion of the probe by the fluorophore could also be important. Overall, we observe nonideal aspects of phospholipid probe mobility and partitioning that may not be restricted to supported bilayers.

Characterizing the interactions between GPI-anchored alkaline phosphatases and membrane domains by AFM

“…The absence of phase separation in Langmuir monolayers experiments might account for the apparent discrepancy between the results. The presence of both line tension between the fluid and ordered domains and packing defects or local heterogeneity in the ordered domains [36,14] likely favors BIAP insertion in both SLBs and biomembranes [75]. Figure 2 is in good accordance with this hypothesis: time dependent experiments showed that the first BIAP dimers were detected at the border of the fluidgel domains.…”

Lipid-Mediated Endocytosis

“…Interaction with cholesterol leads to short-lived confinement in the nanometer range (Spiegel et al 1984;Eggeling et al 2009;Sahl et al 2010). The diffusion of GM1-bound CTX is more than an order of magnitude slower than that of free GM1 in supported membrane bilayers, plasma membrane vesicles, and cellular membranes (Spiegel et al 1984;Kenworthy et al 2004;Burns et al 2005;Lingwood et al 2008;Eggeling et al 2009). The lateral mobility of GSL-bound polyomaviridae is even lower Kukura et al 2009).…”

Enzymatic generation of ceramide induces membrane restructuring: Correlated AFM and fluorescence imaging of supported bilayers

“…As noted recently, it is difficult to find a dye-labeled lipid that will reliably partition into liquid-ordered domains and is compatible with excitation at >480 nm (Baumgart et al, 2007). However, GM1 has been shown to localize in the liquid-ordered phase for a variety of ternary lipid mixtures and can be easily detected by labeling with cholera toxin (Burns et al, 2005;Chiantia et al, 2008;Hammond et al, 2005;Shaw et al, 2006;Yuan et al, 2002). We prepared bilayers from DOPC/ESM/cholesterol vesicles containing 1% GM1 and used binding of Alexa 647 labeled cholera toxin subunit B (Ctx-B) to visualize the protein-labeled ganglioside by both fluorescence and AFM.…”