Session 4: Technology: what do we need to know and how can we measure it?
What can single-molecule observations of protein dynamics in living cells tell us about the organization, function and activity of membrane domains?
Aki Kusumi
The plasma membrane is a non-ideal fluid mixture of a great number of molecules with varying degrees of mutual miscibilities, and is therefore considered to contain various transient molecular complexes and microdomains. Furthermore, a higher level of diffusion control - that is, partitioning of the cell membrane by actin-based membrane skeleton 'fences' and their associated transmembrane protein 'pickets' into compartments that vary in diameter between 30-230 nm (depending on the cell type) - may be at work throughout the cell membrane. Virtually all membrane molecules undergo short-term confined diffusion within a membrane compartment and long-term hop diffusion between compartments, making the diffusion coefficient strongly dependent on the observation time window (unlike simple Brownian diffusion). Given such a complex environment, to understand how molecules encounter each other, or how they are recruited to specific places in the cell membrane, single-molecule methods that can circumvent ensemble averaging will greatly help researchers.
With the partitioning of the plasma membrane by the membrane skeleton fences and pickets, the macroscopic diffusion coefficient becomes highly sensitive to the size of the diffusing unit1,2, and is therefore greatly decreased upon the formation of molecular complexes, clusters and stable raft domains, contrary to previous predictions3 based on the two-dimensional fluid continuum4. The fluid continuum model is perfectly fine, as long as the space scale of the cellular event of interest does not exceed 10 nm (the size of the original figure showing the fluid mosaic model by Singer and Nicholson4). However, the original fluid-mosaic model cannot be applied to events occurring in ranges greater than 10 nm. The dependence of the diffusion rate on the diffusing unit size could be used for the detection of raft formation/stabilization and for the determination of the size and the lifetime of the raft.
In single-molecule tracking in an inhomogeneous medium, frame exposure time and frame acquisition frequency (instrumental response time and the density of the data points) are important experimental parameters (again unlike simple Brownian diffusion). Furthermore, the time resolution must be at least an order of magnitude shorter than the lifetime of the event being observed or, for domain detection, than the passage of time of the molecule in the domain, t = <x2>/ 4D, which may be of the order of 100 �s for domains with a diameter of ~35 nm with D (diffusion coefficient) of 3 �m�/s.
One of the major sources of the great confusions in lipid raft research at present might be a lack of appreciation of the spatiotemporal limitations of the technologies employed by raft researchers, and consequent over-interpretations of the measurements. The raft domains may vary greatly in size and lifetime (as well as molecular composition), and each method used may have been sensitive to only a small subset of rafts present in the cell membrane5.
References
1. Iino, R., Koyama, I. & Kusumi, A. Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface. Biophys. J. 80, 2667-2677 (2001)
2. Murase, K. et al. Ultrafine membrane compartments for molecular diffusion as revealed by single molecule techniques. Biophys. J. 86, 4075-4093 (2004)
3. Saffman, P. G. & Delbr�ck, M. Brownian motion in biological membranes. Proc. Natl Acad. Sci. USA 72, 3111-3113 (1975)
4. Singer, S. J. & Nicolson, G. L. The fluid mosaic model of the structure of cell membranes. Science 175, 720-731 (1972)
5. Kusumi, A., Koyama-Honda, I. & Suzuki, K. Molecular dynamics and interactions for creation of stimulation-induced stabilized rafts from small unstable steady-state rafts. Traffic 5, 213-230 (2004)
| |
| |
Full list of key questions