No abstract

Techniques such as single-particle tracking allow the characterization of the movements of single or very few molecules. Features of the molecular trajectories, such as confined diffusion or directed transport, can reveal interesting biological interactions, but they can also arise from simple Brownian motion. Careful analysis of the data, therefore, is necessary to identify interesting effects from pure random movements. A method was developed to detect temporary confinement in the trajectories of membrane proteins that cannot be accounted for by Brownian motion. This analysis was applied to trajectories of two lipid-linked members of the immunoglobulin superfamily, Thy-1 and a neural cell adhesion molecule (NCAM 125), and the results were compared with those for simulated random walks. Approximately 28% of the trajectories for both proteins exhibited periods of transient confinement, which were < 0.07% likely to arise from random movements. In contrast to these results, only 1.5% of the simulated trajectories showed confined periods. Transient confinement for both proteins lasted on average 8 s in regions that were approximately 280 nm in diameter.

Glycosylphosphatidylinositol (GPI)-anchored proteins participate in many cell surface functions; however, the molecular associations of these lipid-linked proteins within the plasma membrane are not well understood. Recent biochemical analyses of detergent insoluble membrane fractions have suggested that GPI-anchored proteins may be associated with glycosphingolipid (GSL)-enriched domains that also contain cholesterol and signaling molecules such as Src family kinases and, in some cases, caveolae. The movements of two components of the putative GSL-enriched domains, Thy-1, a GPI-anchored protein, and GM1, a GSL, were followed with single particle tracking on C3H 10T1/2 cell surfaces and categorized into four modes of lateral transport, fast diffusion, slow anomalous diffusion, diffusion confined to 325-370 nm diameter regions, and a fraction of molecules that was essentially stationary on the 6.6 s time scale. Longer observations (60 s) showed that Thy-1 and GM1 are transiently confined for 7-9 s to regions averaging 260-330 nm in diameter. Approximately 35-37% of both Thy-1 and GM1 undergo confined diffusion, whereas only 16% of fluorescein phosphatidylethanolamine, a phospholipid analog which is not expected to be found in the GSL domains, experience confined diffusion to regions averaging approximately 230 nm in diameter. Further, when glycosphingolipid expression was reduced approximately 40% with the glucosylceramide synthase inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, the percentage of trajectories exhibiting confinement and the size of the confining domain for Thy-1 were reduced approximately 1.5-fold. In contrast, extraction of cells with Triton X-100 leaves the fraction of molecules confined and the domain sizes of Thy-1 and GM1 unchanged. Our results are consistent with the preferential association of GPI-anchored proteins with glycosphingolipid-enriched domains and suggest that the confining domains may be the in vivo equivalent of the detergent insoluble membrane fractions.

We have employed an interferometric technique for the local measurement of bending modulus, membrane tension, and adhesion energy of motile cells adhering to a substrate. Wild-type and mutant cells of Dictyostelium discoideum were incubated in a flow chamber. The flow-induced deformation of a cell near its adhesion area was determined by quantitative reflection interference contrast microscopy (RICM) and analyzed in terms of the elastic boundary conditions: equilibrium of tensions and bending moments at the contact line. This technique was employed to quantify changes caused by the lack of talin, a protein that couples the actin network to the plasma membrane, or by the lack of cortexillin I or II, two isoforms of the actin-bundling protein cortexillin. Cells lacking either cortexillin I or II exhibited reduced bending moduli of 95 and 160 k(B)T, respectively, as compared to 390 k(B)T, obtained for wild-type cells. No significant difference was found for the adhesion energies of wild-type and cortexillin mutant cells. In cells lacking talin, not only a strongly reduced bending modulus of 70 k(B)T, but also a low adhesion energy one-fourth of that in wild-type cells was measured.

The lateral mobility of the neural cell adhesion molecule (NCAM) was examined using single particle tracking (SPT). Various isoforms of human NCAM, differing in their ectodomain, their membrane anchorage mode, or the size of their cytoplasmic domain, were expressed in National Institutes of Health 3T3 cells and C2C12 muscle cells. On a 6.6-s time scale, SPT measurements on both transmembrane and glycosylphosphatidylinositol (GPI) anchored isoforms of NCAM expressed in 3T3 cells could be classified into mobile (Brownian diffusion), slow diffusion, corralled diffusion, and immobile subpopulations. On a 90-s time scale, SPT studies in C2C12 cells revealed that 40-60% of transfected NCAM was mobile, whereas a smaller fraction (approximately 10-30%) experienced much slower diffusion. In addition, a fraction of approximately 30% of both transfected GPI and transmembrane isoforms and endogenous NCAM isoforms in C2C12 cells experienced transient confinement for approximately 8 s within regions of approximately 300-nm diameter. Diffusion within both these and the slow diffusion regions was anomalous, consistent with movements through a dense field of obstacles, whereas diffusion outside these regions was normal. Thus the membrane appears as a mosaic containing regions that permit free diffusion as well as regions in which NCAM is transiently confined to small or more extended domains. These results, including a large, freely diffusing fraction, similar confinement of transmembrane and GPI isoforms, a significant slowly diffusing fraction, and relatively large interdomain distances, are at some variance with the membrane skeleton fence model (Kusumi and Sako, 1996). Possible revisions to the model that incorporate these data are discussed.

We used micropipettes to aspirate leading and trailing edges of wild-type and mutant cells of Dictyostelium discoideum. Mutants were lacking either myosin II or talin, or both proteins simultaneously. Talin is a plasma membrane-associated protein important for the coupling between membrane and actin cortex, whereas myosin II is a cytoplasmic motor protein essential for the locomotion of Dictyostelium cells. Aspiration into the pipette occurred above a threshold pressure only. For all cells containing talin this threshold was significantly lower at the leading edge of an advancing cell as compared to its rear end, whereas we found no such difference in cells lacking talin. Wild-type and talin-deficient cells were able to retract from the pipette against an applied suction pressure. In these cells, retraction was preceded by an accumulation of myosin II in the tip of the aspirated cell lobe. Mutants lacking myosin II could not retract, even if the suction pressures were removed after aspiration. We interpreted the initial instability and the subsequent plastic deformation of the cell surface during aspiration in terms of a fracture between the cell plasma membrane and the cell body, which may involve destruction of part of the cortex. Models are presented that characterize the coupling strength between membrane and cell body by a surface energy sigma. We find sigma approximately 0.6(1.6) mJ/m(2) at the leading (trailing) edge of wild-type cells.

We report the deformation and unbinding of weakly adhering giant vesicles under hydrodynamic shear forces. Linear shear fields are generated in a flat cell and vesicle adhesion onto a supported membrane is generated by electrostatic forces between oppositely charged lipids. The hydrodynamic flow in the aqueous medium near the outer side of the vesicle, within the vesicle and the tanktreading motion in the membrane are observed by tracing small markers attached to the vesicles or suspended in the medium by confocal laser scanning microscopy. The lift force generated by the rotational flow in the vesicle was estimated to be at least two orders of magnitude larger than predicted by the simple theory.

We present a theory showing that local shape instabilities of composite biological membranes, consisting of a lipid bilayer and an underlying actin cortex, can be triggered by a local softening of the membrane-associated cytoskeleton. A membrane containing such cortical defects can form blisters or invaginations, depending on external conditions. The theoretical predictions agree with observations provided by two sets of experiments: (i) microscopic observations of shape changes of giant vesicles with underlying shells of a thin actin network show the formation of local blisters and (ii) micropipet aspiration experiments of Dictyostelium discoideum cells in which we observed the formation of blisters in the aspirated cell part. In the latter experiments, the existence of a hole in the underlying cortex is confirmed by observation of the entrance of cell organelles into the blister. Our model may also be applied to the formation of lobopodia, fast-growing cell protrusions that play an important role in the locomotion and spreading of biological cells.