Diffusion, Transport, and Cell Membrane Organization Investigated by Imaging Fluorescence Cross-Correlation Spectroscopy

Abstract

Cell membrane organization is dynamic and is assumed to have different characteristic length scales. These length scales, which are influenced by lipid and protein composition as well as by the cytoskeleton, can range from below the optical resolution limit (as with rafts or microdomains) to far above the resolution limit (as with capping phenomena or the formation of lipid "platforms"). The measurement of these membrane features poses a significant problem because membrane dynamics are on the millisecond timescale and are thus beyond the time resolution of conventional imaging approaches. Fluorescence correlation spectroscopy (FCS), a widely used spectroscopic technique to measure membrane dynamics, has the required time resolution but lacks imaging capabilities. A promising solution is the recently introduced method known as imaging total internal reflection (ITIR)-FCS, which can probe diffusion phenomena in lipid membranes with good temporal and spatial resolution. In this work, we extend ITIR-FCS to perform ITIR fluorescence cross-correlation spectroscopy (ITIR-FCCS) between pixel areas of arbitrary shape and derive a generalized expression that is applicable to active transport and diffusion. ITIR-FCCS is applied to model systems exhibiting diffusion, active transport, or a combination of the two. To demonstrate its applicability to live cells, we observe the diffusion of a marker, the sphingolipid-binding domain (SBD) derived from the amyloid peptide Abeta, on live neuroblastoma cells. We investigate the organization and dynamics of SBD-bound lipid microdomains under the conditions of cholesterol removal and cytoskeleton disruption.

The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

“…The plug-in is analogous to our previously described program, ImFCS (49). The correlation functions were fitted with a model for 2D diffusion derived previously in Sankaran et al (50) to extract values of apparent particle numbers N, diffusion coefficient D, and asymptotic correlation G inf . The size of the microscope point spread function was calibrated by measurement in supported lipid bilayers as described in Bag et al (51).…”

Transient State Monitoring by Total Internal Reflection Fluorescence Microscopy

“…28 TIR-mediated evanescent field excitation, combined with a highly sensitive fluorescence detection by an electron multiplied charge-coupled device (EM-CCD) camera has been shown to allow both autocorrelation, 29,30 and more recently also cross-correlation fluctuation analyses of molecular diffusion and transport in molecular membranes. 31 However, while sufficient for relatively slow dynamics, transient state fluctuations typically take place in the µs time range and require a time resolution far beyond that of an EMCCD camera. Such a time resolution in combination with high detection sensitivity allows up to date only a limited number of spots to be measured simultaneously.…”

Plasma membrane reorganization: A glycolipid gateway for microbes

“…Membrane blebs and artificial membranes that lack actin have a higher rate of protein and lipid diffusion when compared to the membranes that contain actin [10]. Recent studies proved that actin depolymerisation prevented the formation of lipid rafts by increasing the diffusion rate of lipid raft markers [11,12]. A multitude of weak binding interactions between cortical actin components and plasma membrane phospholipids provide the adhesion energy required to keep the membrane tethered to the cytoskeletal surface [13].…”