Ultrafine Membrane Compartments for Molecular Diffusion as Revealed by Single Molecule Techniques

Abstract

Plasma membrane compartments, delimited by transmembrane proteins anchored to the membrane skeleton (anchored-protein picket model), would provide the membrane with fundamental mosaicism because they would affect the movement of practically all molecules incorporated in the cell membrane. Understanding such basic compartmentalized structures of the cell membrane is critical for further studies of a variety of membrane functions. Here, using both high temporal-resolution single particle tracking and single fluorescent molecule video imaging of an unsaturated phospholipid, DOPE, we found that plasma membrane compartments generally exist in various cell types, including CHO, HEPA-OVA, PtK2, FRSK, HEK293, HeLa, T24 (ECV304), and NRK cells. The compartment size varies from 30 to 230 nm, whereas the average hop rate of DOPE crossing the boundaries between two adjacent compartments ranges between 1 and 17 ms. The probability of passing a compartment barrier when DOPE is already at the boundary is also cell-type dependent, with an overall variation by a factor of approximately 7. These results strongly indicate the necessity for the paradigm shift of the concept on the plasma membrane: from the two-dimensional fluid continuum model to the compartmentalized membrane model in which its constituent molecules undergo hop diffusion over the compartments.

What do diffusion measurements tell us about membrane compartmentalisation? Emergence of the role of interprotein interactions

“…Moreover, in different systems using different tracking techniques, similar observations were made [50][51][52][53]. Frick et al [54] have recently given additional evidence supporting the previous interpretation by using FRAP on four different proteins.…”

“…As a consequence, assuming that the number of proteins within an assembly is constant, both the microscopic and macroscopic diffusion coefficients remain proportional to L 2 [15]. 40 J Chem Biol (2008) 1:37-48 Moreover, in different systems using different tracking techniques, similar observations were made [50][51][52][53]. Frick et al [54] have recently given additional evidence supporting the previous interpretation by using FRAP on four different proteins.…”

Lipid rafts: contentious only from simplistic standpoints

“…• Kusumi and colleagues have shown that the plasma membrane is compartmentalized by a picket fence of transmembrane proteins that are anchored to the submembrane actin cytoskeleton 26,[67][68][69] . The fence allows relative free lateral diffusion of lipids within compartments but restricts movement between compartments.…”

3D Particle Tracking on a Two-Photon Microscope

“…Five and 2 µm signal were separately input into the piezo, and the recovered motions were within 4% of expected value. 4.13E−10, 4.33E−10, and 4.53E−10 cm 2 /s, for the individual x, y, and z axes, respectively and 4.30E−10 cm 2 /s for the overall diffusion coefficient. Figure 4c and d shows an example of diffusion with flow.…”

“…The technique was extended by using video microscopy and small gold particles (20-40 nm) as the label for the particle of interest [2]. Other groups investigated lipid motion using a high speed video camera and obtained a frequency response of 40 kHz and a precision of 17 nm [3,4]. Kis-Petikova developed a technique to track particles in 2D using the two-photon microscope [5].…”