Mobility and diffusion in the plane of cell membrane

The mobility and diffusion of integral proteins in the plane of the membrane are studied in the light of recently known hydrodynamic equations of liquid crystals. We derive the formulas of mobility and diffusion constant in a two-dimensional liquid crystal, and give the solution of the diffusion equation in a spherical membrane. We then discuss the results of the Frye-Edidin (1970) experiments and compare the data with our theoretical values. We conclude that the (human and mouse cell) membranes of the Frye-Edidin experiments are in a phase transition or critical region at 37 °C. Further experiments are proposed to test our assertion.     

Phospholipids undergo hop diffusion in compartmentalized cell membrane

The diffusion rate of lipids in the cell membrane is reduced by a factor of 5-100 from that in artificial bilayers. This slowing mechanism has puzzled cell biologists for the last 25 yr. Here we address this issue by studying the movement of unsaturated phospholipids in rat kidney fibroblasts at the single molecule level at the temporal resolution of 25 micros. The cell membrane was found to be compartmentalized: phospholipids are confined within 230-nm-diameter (phi) compartments for 11 ms on average before hopping to adjacent compartments. These 230-nm compartments exist within greater 750-nm-phi compartments where these phospholipids are confined for 0.33 s on average. The diffusion rate within 230-nm compartments is 5.4 microm2/s, which is nearly as fast as that in large unilamellar vesicles, indicating that the diffusion in the cell membrane is reduced not because diffusion per se is slow, but because the cell membrane is compartmentalized with regard to lateral diffusion of phospholipids. Such compartmentalization depends on the actin-based membrane skeleton, but not on the extracellular matrix, extracellular domains of membrane proteins, or cholesterol-enriched rafts. We propose that various transmembrane proteins anchored to the actin-based membrane skeleton meshwork act as rows of pickets that temporarily confine phospholipids.     

Polysialylation increases lateral diffusion of neural cell adhesion molecule in the cell membrane

Polysialic acid (PSA) is a polymer of N-acetylneuraminic acid residues added post-translationally to the membrane-bound neural cell adhesion molecule (NCAM). The large excluded volume created by PSA polymer is thought to facilitate cell migration by decreasing cell adhesion. Here we used live cell imaging (spot fluorescence recovery after photobleaching and fluorescence correlation spectroscopy) combined with biochemical approaches in an attempt to uncover a link between cell motility and the impact of polysialylation on NCAM dynamics. We show that PSA regulates specifically NCAM lateral diffusion and this is dependent on the integrity of the cytoskeleton. However, whereas the glial-derivative neurotrophic factor chemotactic effect is dependent on PSA, the molecular dynamics of PSA-NCAM is not directly affected by glial-derivative neurotrophic factor. These findings reveal a new intrinsic mechanism by which polysialylation regulates NCAM dynamics and thereby a biological function like cell migration.     

Detection of non-Brownian diffusion in the cell membrane in single molecule tracking

Molecules undergo non-Brownian diffusion in the plasma membrane, but the mechanism behind this anomalous diffusion is controversial. To characterize the anomalous diffusion in the complex system of the plasma membrane and to understand its underlying mechanism, single-molecule/particle methods that allow researchers to avoid ensemble averaging have turned out to be highly effective. However, the intrinsic problems of time-averaging (resolution) and the frequency of the observations have not been explored. These would not matter for the observations of simple Brownian particles, but they do strongly affect the observation of molecules undergoing anomalous diffusion. We examined these effects on the apparent motion of molecules undergoing simple, totally confined, or hop diffusion, using Monte Carlo simulations of particles undergoing short-term confined diffusion within a compartment and long-term hop diffusion between these compartments, explicitly including the effects of time-averaging during a single frame of the camera (exposure time) and the frequency of observations (frame rate). The intricate relationships of these time-related experimental parameters with the intrinsic diffusion parameters have been clarified, which indicated that by systematically varying the frame time and rate, the anomalous diffusion can be clearly detected and characterized. Based on these results, single-particle tracking of transferrin receptor in the plasma membrane of live PtK2 cells were carried out, varying the frame time between 0.025 and 33 ms (0.03-40 kHz), which revealed the hop diffusion of the receptor between 47-nm (average) compartments with an average residency time of 1.7 ms, with the aid of single fluorescent-molecule video imaging.     

The determination of local cell membrane diffusion coefficients

Solutions applicable to the measurement of cell membrane diffusion coefficients are derived using an approximate integral method.     

Band-3 protein-mediated anion conductance of the red cell membrane. Slippage vs ionic diffusion

The band 3 protein-mediated, valinomycin-induced KCl efflux continues to increase with increasing [KCl] when the Cl-/Cl- equilibrium exchange becomes saturated. This suggests the existence of a band 3-mediated component of Cl- flux that contributes to the electrical conductance without being related to slippage; i.e., equilibration of the unloaded transport protein between the two membrane surfaces.     

Myoglobin and hemoglobin rotational diffusion in the cell.

The detection of the 1H NMR signal of myoglobin (Mb) in tissue opens an opportunity to examine its cellular diffusion property, which is central to its purported role in facilitating oxygen transport. In perfused myocardium the field-dependent transverse relaxation analysis of the deoxy Mb proximal histidyl NdeltaH indicates that the Mb rotational correlation time in the cell is only approximately 1.4 times longer than it is in solution. Such a mobility is consistent with the theory that Mb facilitates oxygen diffusion from the sarcoplasm to the mitochondria. The microviscosities of the erythrocyte and myocyte environment are different. The hemoglobin (Hb) rotational correlation time is 2.2 longer in the cell than in solution. Because both the overlapping Hb and Mb signals are visible in vivo, a relaxation-based NMR strategy has been developed to discriminate between them.     

Heterogeneous nanoscopic lipid diffusion in the live cell membrane and its dependency on cholesterol

Cholesterol plays a unique role in the regulation of membrane organization and dynamics by modulating the membrane phase transition at the nanoscale. Unfortunately, due to their small sizes and dynamic nature, the effects of cholesterol-mediated membrane nanodomains on membrane dynamics remain elusive. Here, using ultrahigh-speed single-molecule tracking with advanced optical microscope techniques, we investigate the diffusive motion of single phospholipids in the live cell plasma membrane at the nanoscale and its dependency on the cholesterol concentration. We find that both saturated and unsaturated phospholipids undergo anomalous subdiffusion on the length scale of 10–100 nm. The diffusion characteristics exhibit considerable variations in space and in time, indicating that the nanoscopic lipid diffusion is highly heterogeneous. Importantly, through the statistical analysis, apparent dual-mobility subdiffusion is observed from the mixed diffusion behaviors. The measured subdiffusion agrees well with the hop diffusion model that represents a diffuser moving in a compartmentalized membrane created by the cytoskeleton meshwork. Cholesterol depletion diminishes the lipid mobility with an apparently smaller compartment size and a stronger confinement strength. Similar results are measured with temperature reduction, suggesting that the more heterogeneous and restricted diffusion is connected to the nanoscopic membrane phase transition. Our conclusion supports the model that cholesterol depletion induces the formation of gel-phase, solid-like membrane nanodomains. These nanodomains undergo restricted diffusion and act as diffusion obstacles to the membrane molecules that are excluded from the nanodomains. This work provides the experimental evidence that the nanoscopic lipid diffusion in the cell plasma membrane is heterogeneous and sensitive to the cholesterol concentration and temperature, shedding new light on the regulation mechanisms of nanoscopic membrane dynamics.     

Lateral electrophoresis and diffusion of Concanavalin A receptors in the membrane of embryonic muscle cell

A uniform electric field of 10 V/cm applied across the surface of embryonic toad Xenopus muscle cells results in the asymmetric accumulation of concanavalin A (Con A) receptors toward one side of the cells within 10 min, as visualized by postfield fluorescent Con A labeling. This field produces an extracellular voltage difference of 20 mV across these 20-microns wide cells. The effect is reversible in two respects: (a) Additional exposure of the cell to the same field of opposite polarity for 10 min completely reverses the asymmetric accumulation to the other side of the cell. (b) Relaxation occurs after the removal of the field and results in complete recovery of the uniform distribution in 30 min. Both the accumulation and the recovery movements are independent of cell metabolism, and appear to be electrophoretic and diffusional in nature. The threshold field required to induce a detectable accumulation by the present method is between 1.0 and 1.5 V/cm (corresponding to a voltage difference of 2-3 mV across a 20-microns wide cell). The electrophoretic mobility of the most mobile population of nonliganded Con A receptors is estimated to be about 2 x 10(-3) microns/s per V/cm, while their diffusion coefficient is in the range of 4-7 x 10(-10) cm2/s. Extensive accumulation of the Con A receptors by an electric field results in the formation of immobile aggregates. The Con A receptors appear to consist of a heterogeneous population of membrane components different in their charge properties, mobility, and capability in forming aggregates.     

Characterization of AtCDC48. Evidence for multiple membrane fusion mechanisms at the plane of cell division in plants

The components of the cellular machinery that accomplish the various complex and dynamic membrane fusion events that occur at the division plane during plant cytokinesis, including assembly of the cell plate, are not fully understood. The most well-characterized component, KNOLLE, a cell plate-specific soluble N-ethylmaleimide-sensitive fusion protein (NSF)-attachment protein receptor (SNARE), is a membrane fusion machine component required for plant cytokinesis. Here, we show the plant ortholog of Cdc48p/p97, AtCDC48, colocalizes at the division plane in dividing Arabidopsis cells with KNOLLE and another SNARE, the plant ortholog of syntaxin 5, SYP31. In contrast to KNOLLE, SYP31 resides in defined punctate membrane structures during interphase and is targeted during cytokinesis to the division plane. In vitro-binding studies demonstrate that AtCDC48 specifically interacts in an ATP-dependent manner with SYP31 but not with KNOLLE. In contrast, we show that KNOLLE assembles in vitro into a large approximately 20S complex in an Sec18p/NSF-dependent manner. These results suggest that there are at least two distinct membrane fusion pathways involving Cdc48p/p97 and Sec18p/NSF that operate at the division plane to mediate plant cytokinesis. Models for the role of AtCDC48 and SYP31 at the division plane will be discussed.     

Passive diffusion of non-electrolytes across the lysosome membrane.

An osmotic-protection method has been used to study the permeability of rat liver lysosomes to 43 organic non-electrolytes of formula weights ranging from 62 to 1000. A lysosome-rich centrifugal fraction of rat liver homogenate was resuspended in an unbuffered 0.25 M solution of test solute, pH 7.0, and incubated at 25 degrees C for 60 min. The free and total activities of 4-methylumbelliferyl N-acetyl-beta-D-glucosaminidase were measured after incubation for 0, 30 and 60 min. Three patterns of results were seen. In pattern A the percentage free activity remained low throughout the 60 min incubation, indicating little or no solute entry into the lysosomes. In pattern B, the percentage free activity was initially low, but rose substantially during the incubation, indicating solute entry. In pattern C there was not even initial osmotic protection, indicating very rapid solute entry. The rapidity of solute entry into the lysosomes showed no correlation with the formula weight, but a perfect inverse correlation with the hydrogen-bonding capacity of the solutes. The results, which can be used to predict the ability of further compounds to cross the lysosome membrane by unassisted diffusion, are discussed in the context of metabolite and drug release from lysosomes in vivo.     

Role of membrane proteins and lipids in water diffusion across red cell membranes

When human red cells are treated with the mercurial sulfhydryl reagent, $\text{p-}\text{chloromercuribenzene sulfonate}$, osmotic water permeability is suppressed and only diffusional water permeability remains (Macey, R.I. and Farmer, R.E.L. (1970) Biochim. Biophys. Acta 211, 104–106). It has been suggested that the route for the remaining water permeation is by diffusion through the membrane lipids. However, after making allowance for the relative lipid area of the membrane, the water diffusion coefficient through lipid bilayers which contain cholesterol is too small by a factor of two or more. We have measured the permeability coefficient of normal human red cells by proton $\text{T}{}_1$ NMR and obtained a value of 4.0 · 10^?3 cm · s^?1, in good agreement with published values. In order to study permeation-through red cell lipids we have perturbed extracted red cell lipids with the lipophilic anesthetic, halothane, and found that halothane increases water permeability. The same concentration of halothane has no effect on the water permeability of human red cells, after maximal pCMBS inhibition. In order to compare halothane mobility in extracted red cell membrane lipids with that in red cell ghost membranes, we have studied halothane quenching of $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ by equilibrium fluorescence and fluorescence lifetime methods. Since halothane mobility is similar in these two preparations, we have concluded that the primary route of water diffusion in pCMBS-treated red cells is not through membrane lipids, but rather through a membrane protein channel.     

Lateral diffusion in the plasma membrane of maize protoplasts with implications for cell culture

Plasma-membrane dynamics in live protoplasts from maize (Zea mays L.) roots were characterized and examined for relationships as to the ability of the protoplasts to synthesize new cell walls and develop to cells capable of division. The lateral diffusion-coefficients and mobile fractions of fluorescence-labeled plasma-membrane proteins and lipids were measured by fluorescence photobleaching recovery. Small but significant effects on the diffusion of membrane proteins were observed after treatments with oryzalin or amiprophosmethyl, microtubule-disrupting drugs that increased the mobile fraction, and after treatments with cytochalasins B or D, microfilament-disrupting drugs that decreased the diffusion coefficient. A number of parameters were tested for correlative effects on membrane dynamics and protoplast performance in culture. Protoplasts isolated with a cellulase preparation from Trichoderma viride showed faster membrane-protein diffusion and a lower frequency of development to cells capable of division than did protoplasts isolated with a cellulase preparation from T. reesei. Membrane proteins in maize A632, a line less capable of plant regeneration from callus, diffused with a smaller diffusion coefficient but a greater mobile fraction than did membrane proteins in maize A634, a line with greater regeneration capacity. The plasma membranes of A632 and A634 protoplasts also differed with regard to lateral-diffusion characteristics of phospholipid and sterol probes, although the presence of both rapidly and slowly diffusing lipid components indicated the apparent existence of lipid domains in both A632 and A634. The protoplasts of the two lines did not differ significantly, however, in either wall regeneration or frequency of development to cells capable of division.Abbreviations and symbols D lateral diffusion coefficient - FITC fluorescein-5-isothiocyanate - FPR fluorescence photobleaching recovery - LY Lucifer yellow - LY-Chol dilithium 4-amino-N-[(-(carbo(5-cholesten-3-yl)oxy)hydrazinocarbonyl)aminol]-1,8-naphthalimide-3,6-disulfonate - LY-DC_16:0PE dilithium 4-amino-N-[3-(-(dipalmitoyl-sn-glycero-3-phosphoethanol-amino)ethylsulfonyl)phenyl]-1,8-naphthalimide-3,6-disulfonate