Fluorescence Corralation Spectroscopy
2005; Elsevier BV; Volume: 89; Issue: 6 Linguagem: Inglês
10.1529/biophysj.105.074161
ISSN1542-0086
Autores Tópico(s)Nanopore and Nanochannel Transport Studies
ResumoIdentifying submicroscopic corrals in the plasma membrane has been of interest since the classic Sheetz barnyard model of confinement of proteins by the erythrocyte membrane skeleton (1). In this issue, Wawrezinieck et al. (2.Wawrezinieck L. Rigneault H. Marguet D. Lenne P.-F. Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization.Biophys. J. 2005; 89: 4029-4042Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar) present a novel fluorescence correlation spectroscopy (FCS) method to identify corrals. What is a corral? From the standpoint of cell biology, there are several possible corrals: a), the spectrin-actin membrane skeleton, a lattice of triangles of side ∼75 nm (1.Sheetz M.P. Membrane skeletal dynamics: Role in modulation of red cell deformability, mobility of transmembrane proteins, and shape.Semin. Hematol. 1983; 20: 175-188PubMed Google Scholar); b), actin filaments near the membrane, possibly with transiently or permanently bound transmembrane proteins forming the pickets of Kusumi's picket fence model, side 32–110 nm (3.Kusumi A. Nakada C. Ritchie K. Murase K. Suzuki K. Murakoshi H. Kasai R.S. Kondo J. Fujiwara T. Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: High-speed single-molecule tracking of membrane molecules.Annu. Rev. Biophys. Biomol. Struct. 2005; 34: 351-378Crossref PubMed Scopus (917) Google Scholar); c), a lipid domain, either raft or nonraft, with the raft size 0–700 nm (4.Anderson R.G.W. Jacobson K. A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains.Science. 2002; 296: 1821-1825Crossref PubMed Scopus (1006) Google Scholar); d), the extracellular matrix (5.Masuda A. Ushida K. Okamoto T. New fluorescence correlation spectroscopy enabling direct observation of spatiotemporal dependence of diffusion constants as an evidence of anomalous transport in extracellular matrices.Biophys. J. 2005; 88: 3584-3591Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar); and e), tethers connecting the mobile species to an immobile structure. To identify a corral from diffusion measurements, a corral must be taken to be an area with boundaries impenetrable enough that a mobile particle is confined to the area for a time much longer than the diffusion time across the area. One must be able to distinguish real confinement from the apparent confinement that is an inherent fluctuation in an unconfined random walk. This statistical problem is discussed in the context of single-particle tracking by Saxton and Jacobson (6.Saxton M.J. Jacobson K. Single-particle tracking: Applications to membrane dynamics.Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 373-399Crossref PubMed Scopus (1469) Google Scholar) and in the supplemental material of Kusumi et al. (3.Kusumi A. Nakada C. Ritchie K. Murase K. Suzuki K. Murakoshi H. Kasai R.S. Kondo J. Fujiwara T. Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: High-speed single-molecule tracking of membrane molecules.Annu. Rev. Biophys. Biomol. Struct. 2005; 34: 351-378Crossref PubMed Scopus (917) Google Scholar). How can a corral be detected? In a fluorescence photobleaching recovery (FPR) experiment, it is necessary to replenish the entire bleached spot to obtain a complete recovery curve. Diffusion is therefore over an area on the order of 10 times the spot size, where the spot size is diffraction-limited or larger. For the membrane skeleton or actin corrals, the measurement thus averages over many corrals and the diffusion coefficient is proportional to the corral area/mean residence time. Lipid domains, however, may be large enough to be detectable. In FPR measurements of lipid and protein diffusion in fibroblasts, Yechiel and Edidin (7.Yechiel E. Edidin M. Micrometer-scale domains in fibroblast plasma membranes.J. Cell Biol. 1987; 105: 755-760Crossref PubMed Scopus (191) Google Scholar) varied the spot size over a range of 0.35–5.0 μm, and found that the mobile fraction decreased monotonically with spot size. They interpreted their results as showing protein-rich domains of radius 0.4–1.5 μm surrounded by a protein-poor continuum. For years, most measurements of lateral diffusion were made by FPR, but a combination of technical advances, reviewed by Webb (8.Webb W.W. Fluorescence correlation spectroscopy: inception, biophysical experimentations, and prospectus.Appl. Opt. 2001; 40: 3969-3983Crossref PubMed Scopus (114) Google Scholar), brought about a renaissance in FCS. It is now highly popular, as a walk through the appropriate poster sessions at the Biophysical Society annual meeting shows. FCS has single-molecule sensitivity, and averaging is carried out to obtain the autocorrelation function, so data analysis is less difficult than in single-particle tracking (reviews: (9.Haustein E. Schwille P. Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy.Methods. 2003; 29: 153-166Crossref PubMed Scopus (207) Google Scholar, 10.Lagerholm B.C. Weinreb G.E. Jacobson K. Thompson N.L. Detecting microdomains in intact cell membranes.Annu. Rev. Phys. Chem. 2005; 56: 309-336Crossref PubMed Scopus (184) Google Scholar, 11.Rigler R. Elson E. Fluorescence Correlation Spectroscopy: Theory and Applications. Springer, Berlin2001Crossref Google Scholar). How can this highly sensitive method be used to identify corrals? The experimental approach, developed independently by Masuda et al. (5.Masuda A. Ushida K. Okamoto T. New fluorescence correlation spectroscopy enabling direct observation of spatiotemporal dependence of diffusion constants as an evidence of anomalous transport in extracellular matrices.Biophys. J. 2005; 88: 3584-3591Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and Wawrezinieck et al. (2.Wawrezinieck L. Rigneault H. Marguet D. Lenne P.-F. Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization.Biophys. J. 2005; 89: 4029-4042Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar), is to vary the beam size. The major advance made by Wawrezinieck et al. (2.Wawrezinieck L. Rigneault H. Marguet D. Lenne P.-F. Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization.Biophys. J. 2005; 89: 4029-4042Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar) is in the method of data analysis. In their FCS measurements and simulations, they varied the beam size w, and measured τ, the width of the autocorrelation function at half-maximum. For free diffusion, τ is linear in the beam area,τ=k1w2,with k1 proportional to 1/D, where D is the diffusion coefficient. But for diffusion in the plasma membrane, they found experimentally, and confirmed by extensive simulations of confined diffusion, that the function is not linear but affine,τ=t0+k2w2,with t0 and k2 constants. Furthermore, the y-intercept t0 is positive for trapping by an isolated corral but negative for trapping in a network of barriers. Plots from the simulations show three regions:Beam area ≪ corral area: free diffusion; linear dependence.Beam area ∼ corral area: transitional region.Beam area ≫ corral area: affine dependence. The beam radius is 190–400 nm, but as the authors point out, the autocorrelation function can be affected by processes on smaller length scales. (Trivially, consider a point high-affinity binding site for the diffusing species.) Their estimate of the minimum detectable microdomain radius is 60 nm. Single-particle tracking gives higher resolution, with particle positions resolved to tens of nanometers (6.Saxton M.J. Jacobson K. Single-particle tracking: Applications to membrane dynamics.Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 373-399Crossref PubMed Scopus (1469) Google Scholar), but the ability to detect 60-nm corrals by FCS is an important advance. The work of Wawrezinieck et al. (2.Wawrezinieck L. Rigneault H. Marguet D. Lenne P.-F. Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization.Biophys. J. 2005; 89: 4029-4042Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar) is a promising beginning. The theory remains to be done, and a test of the method and the resolution in a well-defined (nanofabricated?) model system. It would be useful for the authors to revisit the experiments of Yechiel and Edidin (7.Yechiel E. Edidin M. Micrometer-scale domains in fibroblast plasma membranes.J. Cell Biol. 1987; 105: 755-760Crossref PubMed Scopus (191) Google Scholar) to see whether the new FCS approach can tell us more about these influential results. Likewise it would be interesting to examine by FCS the larger corrals found in the single-particle tracking experiments of the Kusumi laboratory (3.Kusumi A. Nakada C. Ritchie K. Murase K. Suzuki K. Murakoshi H. Kasai R.S. Kondo J. Fujiwara T. Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: High-speed single-molecule tracking of membrane molecules.Annu. Rev. Biophys. Biomol. Struct. 2005; 34: 351-378Crossref PubMed Scopus (917) Google Scholar). The most informative experiments would use the same cell types, diffusing species, and labels as the earlier measurements.
Referência(s)