Lateral Diffusion Of Proteins Research Articles

Apical localization of pair-rule transcripts requires 3′ sequences and limits protein diffusion in the Drosophila blastoderm embryo

The peripheral cytoplasm (periplasm) of the Drosophila blastoderm embryo is subdivided into apical and basal compartments by a layer of nuclei. We have demonstrated three classes of periplasmic transcript localization: apical, basal, and unlocalized (apical and basal), each of which depends on 3′ sequences. We define 3′ apical localization signals within the even-skipped, fushi tarazu, and hairy pair-rule segmentation genes and the α 1-tubulin and bicoid genes. 3′ human α-globin sequences direct transcripts basally. Transcript destination depends on transcript structure, not on transcript stability or chromosomal location. Apical transcripts direct apical compartmentalization of cytoplasmic protein. We propose that apical localization of pair-rule transcripts restricts lateral protein diffusion, thereby allowing pair-rule proteins to define sharp boundaries and precise spatial domains.

Acute toxicity effects of mercury and other heavy metals on HeLa cells and human lymphocytes evaluated via microcalorimetry

We have developed a microcalorimetric bioassay for acute toxicity based on heat evolution from intact cells. A reduction in heat production by cells reflects the inhibition of various biochemical reactions which constitute the cell's metabolic processes. For ten toxicants, including the heavy metals Hg, Cd, Cu, Pb, Cr, V, and As, we have measured the concentrations that are 50% effective (EC 50) in inhibiting heat evolution in tissue-cultured HeLa cells and in human peripheral blood lymphocytes. These data are compared with results of other acute toxicity tests and with microcalorimetric studies of heat evolution in the chemiluminescent bacterium Photobacterium phosphoreum. This comparison indicates that heat production may be the optimum measure of cellular metabolism for instrumental toxicological purposes since it reflects the sum of cellular metabolic processes and since it detects diffferential sensitivities of various cell types to toxicants. We have further studied effects of one heavy metal, mercury, on cell plasma membrane proteins using fluorescence photobleaching recovery (FPR) methods. Mercury induces formation of large membrane protein aggregates which disrupt cell function. FPR results suggest that, even at very low Hg concentrations, small aggregates form rapidly and increase in size causing, within one hour, measureable reductions in protein lateral diffusion.

The membrane skeleton of erythrocytes. A percolation model

The spectrin network on the cytoplasmic surface of the erythrocyte membrane is modeled as a triangular lattice of spectrin tetramers. This network obstructs lateral diffusion of proteins and provides mechanical reinforcement to the membrane. These effects are treated in a systematic and unified manner in terms of a percolation model. The diffusion coefficient is obtained as a function of the fraction of normal spectrin tetramers for both static and fluctuating barriers. The elasticity of the network is calculated as a function of the fraction of normal spectrin and the ratio of bending to stretching energies. For static barriers, elasticity and lateral diffusion are incompatible: if a network is connected enough to be elastic, it is connected enough to block long-range lateral diffusion. The elasticity and the force required for mechanical breakdown go to zero at the percolation threshold; experimental evidence suggests the existence of a stability threshold at or near the percolation threshold. The model is qualitatively applicable to other cells with membrane skeletons, such as epithelial cells, in which localization of membrane proteins is essential to differentiation.

Stabilization of states I and II by p-benzoquinone treatment of intact cells of Chlamydomonas reinhardtii

Intact cells of the unicellular green alga C. reinhardtii were treated with p-benzoquinone after adaptation to state I by far-red illumination or to state II by an incubation under anaerobic conditions. This reagent can be regarded both as an oxidant of the plastoquinones and a protein cross-linker. When algae placed in state I then treated with p-benzoquinone were transferred to state II conditions, and conversely, they retained the fluorescence yield characteristic of their initial state. We could thus obtain intact cells blocked in either state I or II with a similar oxidation state of the electron transfer chain. Analysis of the concentration dependences for state fixation by p-benzoquinone indicated that its reduction prevented state fixation, as expected from a cross-linking action mechanism. We present evidence for such a cross-linking action at the thylakoid membrane level. However, we could rule out the possibility that state fixation occurred through LHC immobilization due to an extensive protein cross-linking, since p-benzoquinone treatment did not prevent protein lateral diffusion along the thylakoid membranes. In contrast, it did prevent changes in LHC phosphorylation. We conclude that the inactivation of the enzymes (kinase and phosphatase) controlling LHC phosphorylation changes is responsible for state fixation. We discuss the mechanism of such enzyme inactivation by p-benzoquinone in terms of intramolecular cross-links.

Distribution and lateral mobility of glycine receptors on cultured spinal cord neurons

Strychnine is one of the most potent antagonists of glycine-mediated inhibitory conductances in the mammalian spinal cord. In order to examine the distribution of glycine receptors (GlyRs) on neuronal cells, 2 novel fluorescent strychnine derivatives were synthesized and characterized chemically, spectroscopically, and biologically. Both compounds retain their biological activity after derivatization and are potent inhibitors of 3H-strychnine binding to bovine spinal cord membranes and membranes from rat spinal cord cultures. Using these fluorescent strychnine analogs, the cellular distribution and lateral mobility of GlyRs on cultured rat spinal cord neurons were studied by digital fluorescence imaging and photobleach recovery microscopy. On these neurons, even in the absence of observable synaptic contact and early in development GlyRs are predominantly localized to cell bodies with sparse labeling of neuritic processes. Although GlyRs are confined to the neuronal cell body, approximately 50% of the receptors are very mobile, with lateral diffusion coefficients of 1.15 +/- 0.05 x 10(-9) cm2/sec, a value which is characteristic of unrestricted protein lateral diffusion. However, the remaining fraction of these receptors are immobile on the neuronal cell body. More than 70% of the GlyRs distributed on neuronal processes are immobile, while 30% are laterally mobile, with diffusion rates of 5.50 +/- 0.1 x 10(-10) cm2/sec. The results indicate that even early in development GlyRs are expressed and segregated to the cell body, where they are confined within a domain that restricts their redistribution.

Lateral diffusion of proteins and lipids in the plasma membrane of rose protoplast

Lateral diffusion measurements have been made on lipids and proteins in the plasma membrane of live protoplasts derived from rose (Rosa sp. “Paul's Scarlet”) suspension-cultured cells. Two different fluorescent lipid probes exhibited markedly different diffusion rates, indicating possible heterogeneity in the lipid domain of the membrane. Membrane proteins were labeled directly with covalently-reactive fluorophores, and factors that might perturb the lateral diffusion of these labeled proteins were investigated. Treatment of the protoplasts with various cytoskeleton-disrupting drugs generally had little effect on protein diffusion, although treatment with oryzalin, a microtubule-disrupting drug, did slightly reduce the mobile fraction of membrane proteins. Elevation of the CaCl2 concentration in the medium from 1 mM to 10 mM significantly reduced the mobile fraction of membrane proteins and also increased the fraction of protoplasts that were able to regenerate cell walls and divide in culture. These results are discussed in relation to reported evidence of lipid domains in the plasma membranes of other cells and protoplasts. The relative importance of lipid domains and membrane-cytoskeleton interaction in governing protein diffusion is considered.

Effect of centrophenoxine and BCE-001 treatment on lateral diffusion of proteins in the hepatocyte plasma membrane as revealed by fluorescence recovery after photobleaching in rat liver smears

The average lateral diffusion coefficient of proteins (D) in the cell membrane of hepatocytes has been measured in liver smears by fluorescence recovery after photobleaching (FRAP), based on the so-called peroxide-induced autofluorescence (PIAF) deriving from the oxidation of riboflavin bound to membrane proteins. It has been previously shown that D displays a significant negative linear age correlation. The in vivo effect of two drugs were tested on this parameter. Young (2.7 months) and old (24–26 months) male rats received centropheroxine (CPH) or a new drug (BCE-001) by either intraperitoneal (i.p.) injection or per os through a gastric tube for 26 to 42 days. D was measured on a double-blind basis in the hepatocyte plasma membrane of treated and control groups. The CPH and BCE-001 treatments did not affect the value of D in the young rats. However, the latter drug increased their growth rate. An increase of D in old animals was induced by treatment with either drug. When the drug effects in old rats were compared, BCE-001 proved to be more efficient than CPH, and at the same time was able to significantly retard the age-dependent loss of body weight characteristic of these animals at the age of approximately 2 years. Our results are in good accord with the predictions of the membrane hypothesis of aging as regards the role of properly placed OH . free radical scavengers in the improvement of membrane and overall cell function.

Alpha interferon accelerates lateral diffusion of Daudi cell surface differentiation antigens: Measurement by fluorescence redistribution after photobleaching

Lateral diffusion coefficients (D) of two surface differentiation antigens (sIgM and Bp35) were determined on interferon-sensitive (-IFs) or resistant (-IFr) Daudi cells by fluorescence photobleaching, using monospecific FITC-anti-IgM or PE-anti-Leu 16 probes. For untreated Daudi -IFs, mean (D) were 5.8 and 5.3 (x10(-10) cm2/sec). These increased, to 11 and 7.9 x 10(-10) cm2/sec (p less than 0.001) within 30 min after binding of recombinant IFN-a (80 to 800 U/10(6) cells), but decreased by up to 4-fold after Con A Mean (D) of identical surface antigens on Daudi-IFr were 8.2 and 9.4 x 10(-10) cm2/sec; and were not altered by IFN-a. Mean (D) of a lipid analog was up to 40-fold higher than for surface proteins and statistically identical in Daudi-IFs and Daudi-IFr. Rapid acceleration by IFN-a of surface protein lateral diffusion in Daudi-IFs obviously could facilitate anti-proliferative signal transduction; by contrast, a baseline increase of (D) in Daudi-IFr was evidently associated with their refractory state.

CAP FORMATION BY PROTEINS ON THE CELL MEMBRANE: EXCLUDED VOLUME EFFECTS

We study the lateral diffusion of proteins in the outer membrane of a biological cell. The “Bretcher” lipid flow field can sweep these particles together into a single cap, provided the flow is strong enough. We analyze the effect of the excluded volume of the proteins on the structure of the cap.

Tumor necrosis factor and immune interferon act in concert to slow the lateral diffusion of proteins and lipids in human endothelial cell membranes.

Vascular endothelial surface-related activities may depend on the lateral mobility of specific cell surface macromolecules. Previous studies have shown that cytokines induce changes in the morphology and surface antigen composition of vascular endothelial cells in vitro and at sites of immune and inflammatory reactions in vivo. The effects of cytokines on membrane dynamic properties have not been examined. In the present study, we have used fluorescence photobleaching recovery (FPR) to quantify the effects of the cytokines tumor necrosis factor (TNF) and immune interferon (IFN-gamma) on the lateral mobilities of class I major histocompatibility complex protein, of an abundant 96,000 Mr mesenchymal cell surface glycoprotein (gp96), and of a phospholipid probe in cultured human endothelial cell (HEC) membranes. Class I protein and gp96 were directly labeled with fluorescein-conjugated monoclonal antibodies; plasma membrane lipid mobility was examined with the phospholipid analogue fluorescein phosphatidylethanolamine (Fl-PE). In untreated, confluent HEC monolayers, diffusion coefficients were 30 x 10(-10) cm2 s-1 for class I protein, 14 x 10(-10) cm2 s-1 for gp96, and 80 x 10(-10) cm2 s-1 for Fl-PE. Fractional mobilities were greater than 80% for each probe. Cultures treated at visual confluence for 3-4 d with either 100 U/ml TNF or 200 U/ml IFN-gamma did not exhibit significant changes in protein or lipid mobilities despite significant changes in cell morphology and membrane antigen composition. In HEC cultures treated concomitantly with TNF and IFN-gamma, however, diffusion coefficients decreased by 71-79% for class I protein, 29-55% for gp96, and 23-38% for Fl-PE. Fractional mobilities were unchanged. By immunoperoxidase transmission electron microscopy, plasma membranes of untreated and cytokine-treated HEC were flat and stained uniformly for class I antigen. "Line" FPR measurements on doubly treated HEC demonstrated isotropic diffusion of class I protein, gp96, and Fl-PE. Finally, although TNF and IFN-gamma retarded the growth of HEC cultures and disrupted the organization of cell monolayers, the slow diffusion rates of gp96 and Fl-PE in confluent doubly treated monolayers were not reproduced in sparse or subconfluent untreated monolayers. We conclude that the slowing of protein and lipid diffusion induced by the combination of TNF and IFN-gamma is not due to plasma membrane corrugations, to anisotropic diffusion barriers, or to decreased numbers of cell-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS)

Cleavage furrow formation of [formula omitted] eggs observed with fluorescence pattern photobleaching

Taking advantage of the extremely slow lateral diffusion of proteins on Rana amurensis egg surface (Xu et al., 1984), it was possible to make a sharp concentric ring pattern on the egg surface by photobleaching of the fluorescein-labelled egg surface. The shape change of the pattern reveals the movement of the egg surface. The surface, even in front of the furrow tip, is drawn towards the furrow centre. The furrow tip differentiates into border and central lines as shown by the distribution of surface protrusions. Between the border and central lines the nascent membrane inserts while the width of furrow tip increases to nearly 100μm.

Lateral diffusion of proteins in membranes.

Membrane protein lateral diffusion can be constrained in several ways: Diffusion can be slower than that predicted for a simple, fluid lipid bilayer; diffusion can be confined to certain regions within the total membrane; and diffusion may not be equally probable in all directions, i.e. it may be anisotropic. We know that protein diffusion is reduced by increasing concentrations of membrane proteins and by interactions of the diffusant with structure(s) peripheral to the membrane. The molecular nature of such peripheral constraints has been difficult to pinpoint, but attention is now being directed to the extracellular matrix in addition to the membrane-associated cytoskeleton. There are many proteins that are confined to lateral domains in differentiated, isolated cells and in cells organized into tissue. The mechanisms that maintain such inhomogeneous distributions should be elucidated in the next few years. Whether lateral diffusion of membrane proteins over distances of a few micrometers is usually isotropic or anisotropic will be ascertained in the near future using imaging methods combined with photobleaching.

Technical improvement of first-dimension slab gel for two-dimensional electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is widely applied for the analysis of complex protein mixtures. Many 2-D PAGE systems and designs are commonly in use. Among these, there is a large group which, for mainly technical reasons, require rod shaped gels for the separation in the first dimension (see e.g. refs. 1-6). Thus, the known advantages of elcctrophorcsis in slab gels. namely sharper and faster resolutions, simultaneous separation of several samples under identical conditions and convenient handling arc not exploited in the first separation step. In this paper, a procedure is described which allows to perform the I -D run in a slab gel. It is demonstrated that the proposed design fits also for those 2-D PAGE systems which normally need rod shaped gels in the first dimension. The widely used slab gel electrophoresis apparatus introduced by Studier [7] can be applied to runs in both dimensions. However, for two reasons a slight modification of the common slab gel cuvette is necessary: (1) Perfectly rectangular stripes containing the separated protein bands have to be cut out of the gel to bc processed for the second run. This operation is not easily carried out, especially when the acrylamide concentration is low (i.e. T = 4%). (2) Considerable lateral diffusion of protein may occur during electrophorcsis in slab gels, especially whcn the protein concentration in a band is high and the adjactent tracks are not used. This may lead to loss of protein when cutting gel stripes of a given size. These problems can bc overcome, when plastic stripes (PVC) are glued (two component adhesive based on epoxy' resin) on that glass plate which normally keeps the slot former, as shown in Fig. l a. The plastic stripes, which are about 0.04 mm thinner than the spacers, serve as excellent guidance when cutting the gel stripes with a razor blade. In addition, lateral diffusion is sufficiently reduced (Fig. 2a, b).

Lateral diffusion of proteins in the periplasm of Escherichia coli.

We have introduced biologically active, fluorescently labeled maltose-binding protein into the periplasmic space of Escherichia coli and measured its lateral diffusion coefficient by the fluorescence photobleaching recovery method. Diffusion of this protein in the periplasm was found to be surprisingly low (lateral diffusion coefficient, 0.9 X 10(-10) cm2 s-1), about 1,000-fold lower than would be expected for diffusion in aqueous medium and almost 100-fold lower than for an equivalent-size protein in the cytoplasm. Galactose-binding protein, myoglobin, and cytochrome c were also introduced into the periplasm and had diffusion coefficients identical to that determined for the maltose-binding protein. For all proteins nearly 100% recovery of fluorescence was obtained after photobleaching, indicating that the periplasm is a single contiguous compartment surrounding the cell. These data have considerable implications for periplasmic structure and for the role of periplasmic proteins in transport and chemotaxis.

Variation in protein lateral diffusion coefficients is related to variation in protein concentration found in mitochondrial inner membranes

The electrophoretic freeze-fracture electron microscopy method (Sowers, A.E. and Hackenbrock, C.R. (1984) Proc. Natl. Acad. Sci. USA 78, 6246–6250) for measuring the lateral diffusion coefficient of integral proteins was applied to a large population of spherical-shaped mitochondrial inner membranes. Membrane integral protein concentration was estimated by determining the intramembrane particle concentration. Analysis of the data reveals that: (a) the radii of the spherical inner membranes in the selected population ranged from 0.22 to 1.2 μm, (b) the intramembrane particle concentrations ranged from 2300 to 6400 per μm 2, and (c) the calculated lateral diffusion coefficients of the intramembrane particles ranged from 1.3·10 −10 to 3.35·10 −9 cm 2/s. The data clearly show a naturally occurring large range in protein concentration in the mitochondrial inner membrane and an inverse correlation of lateral diffusion coefficient with the membrane protein concentration. This study is the first to show that the lateral diffusion coefficient of integral proteins in a native membrane varies as the membrane protein concentration.

Protein lateral movement in lipid bilayers. Stimulation studies of its dependence upon protein concentration

A number of mechanisms have been proposed to account for the decrease in protein lateral diffusion coefficients in a lipid bilayer membrane, as the concentration of proteins is increased. One such mechanism is the steric hindrance (via, say, a hard-core repulsion) to the lateral movement of a protein due to the proximity of other proteins. Here a model is presented to study this effect alone. It is argued that the model will overestimate the effect being studied. The results of computer simulations show that such a mechanism will decrease the lateral diffusion coefficient by less than a factor of 20 below the zero-concentration limit, even when up to 81.7% of the bilayer surface is composed of integral proteins. This result supports the opinion (Kell, D.B. (1984) Trends Biochem. Sci. 9, 379) that such a mechanism cannot account for a decrease in the lateral diffusion coefficient by two or three orders of magnitude.

Polymeric phospholipids as new biomaterials.

Phospholipid polymers form a new class of biomaterials with many potential applications in medicine and research. The development of these compounds is based upon the mimicry of cell surfaces and reflects our current understanding of the properties of membrane lipids. Physicochemical characterization of the monomeric, diacetylenic phospholipids illustrates the similarities to naturally occurring lipids, similarities that are confirmed by the capacity to enrich the membranes of A. laidlawii to the level of 90% diacetylenic lipid. Polymerization of diacetylenic phospholipids is easily attained by irradiation and produces a stable, crystalline array. The ability to link membrane lipids covalently permits the isothermal restriction in their motion, and is useful in basic studies of biomembranes. The thromboresistance of polymeric phosphatidylcholines in vitro may be a consequence of the inability of phosphatidylcholines to participate in coagulation. The restricted lateral diffusion of proteins along a polymeric lattice will also inhibit the formation of coagulation complexes. Existing polymers may be altered by a coating of polymeric lipid obtained by the Langmuir-Blodgett method. Polymerized vesicles display significant reductions in permeability and aggregation. Entrapment of soluble materials and reconstitution of membrane proteins may be exploited in controlled and site-directed drug delivery. Polymerization of cells in situ produces "cellular capsules" with entrapped membrane and cellular components. Polymeric hemosomes are capable of gas transport and may function as red cell surrogates. The hybrid qualities of biomembranes (polar surfaces, nonthrombogenic, low antigenic potential, and low permeability) and synthetic polymers (chemical and physical stability) suggest that polymeric phosphatidylcholines may serve as models for biomaterials design.

Lateral and rotational diffusion of bacteriorhodopsin in lipid bilayers: experimental test of the Saffman-Delbrück equations.

Lateral diffusion of bacteriorhodopsin and a lipid analogue has been measured in dimyristoylphosphatidylcholine bilayers as a function of temperature, phospholipid/protein (mol/mol; L/P) ratio, and aqueous phase viscosity. The protein lateral diffusion coefficients measured above the temperature at which the lipid gel-liquid/crystalline phase transition occurs (Tc) are combined with previously determined rotational diffusion coefficients to provide a test of the Saffman-Delbrück equations [Saffman, P. G. & Delbrück, M. (1975) Proc. Natl. Acad. Sci. USA 72, 3111-3113]. Insertion of the diffusion coefficients into these equations enables the protein diameter to be calculated. The value of 4.3 +/- 0.5 nm so obtained is in reasonable agreement with the known structure of bacteriorhodopsin. A 12-fold increase in the viscosity of the aqueous phase reduces protein lateral diffusion coefficients by 50%, which is also consistent with the Saffman-Delbrück equations. Both protein and lipid lateral diffusion coefficients decrease with decreasing L/P ratio above the Tc. It is argued that, at a high L/P ratio, this effect is probably due to changes in membrane viscosity while, at a low L/P ratio, "crowding" effects (steric restrictions) and protein aggregation become important. When comparing diffusion measurements made in different systems, it is important to take the effect of the L/P ratio into account. When this is done, other published measurements of freely diffusing membrane proteins are in good agreement with the present results and the predictions of the Saffman-Delbrück equations. Below the Tc, the presence of protein enhances diffusion rates. The overall effect is to smooth out the large change in diffusion coefficient that occurs at the Tc.

Changes in the properties of the surface membrane of schistosoma mansoni during growth as measured by fluorescence recovery after photobleaching

Schistosomiasis is a widespread tropical disease of human beings. Infection occurs when a free-living, water-borne cercaria penetrates the skin, becomes transformed into a schistosomulum which then migrates through the blood stream of the host to develop into an adult parasite in the portal blood sys- tem [ 11. The surface membrane of both the schisto- somulum and adult worm is unusual; it is a double bilayer, and forms a continuous surface covering the peripheral syncytium [2]. The properties of this membrane change during growth and development in the host so that it becomes progressively less suscepti- ble to damage by a variety of agents [3-81. An under- standing of the nature of these changes is sought in the hope that immunological control or chemotherapy of the disease may be made more effective. The tech- nique of fluorescence recovery after photobleaching (FRAP) can measure the lateral diffusion of protein and lipid in the cell surface [9-131. We describe here its use in comparing the outer surfaces of the schisto- somulum and adult

Lateral diffusion of gramicidin S, M-13 coat protein and glycophorin in bilayers of saturated phospholipids: Mean field and Monte Carlo studies

We have developed a general model that relates the lateral diffusion coefficient of one isolated large intrinsic molecule (mol. wt. ≳1000) in a phosphatidylcholine bilayer to the static lipid hydrocarbon chain order. We have studied how protein lateral diffusion can depend upon protein-lipid interactions but have not investigated possible non-specific contributions from gel-state lattice defects. The model has been used in Monte Carlo simulations or in mean-field approximations to study the lateral diffusion coefficients of Gramicidin S, the M-13 coat protein and glycophorin in dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC) bilayers as functions of temperature. Our calculated lateral diffusion coefficients for Gramicidin S and the M-13 coat protein are in good agreement with what has been observed and suggest that Gramicidin S is in a dimeric form in DMPC bilayers. In the case of glycophorin we find that the ‘ice breaker’ effect can be understood as a consequence of perturbation of the lipid polar region around the protein. In order to understand this effect is is necessary that the protein hydrophilic section perturb the polar regions of at least approx. 24 lipid molecules, in good agreement with the numbers of 29–30 measured using 31P-NMR. Because of lipid-lipid interactions this effect extends itself out to four or five lipid layers away from the protein so that the hydrocarbon chains of between approx. 74 and approx. 108 lipid molecules are more disordered in the gel phase, so contributing less to the transition enthalpy, in agreement with the numbers of 80–100 deduced from differential scanning calorimetry (DSC). An understanding of the abrupt change in the diffusion coefficient at a temperature below the main bilayer transition temperature requires an additional mechanism. We propose that this change may be a consequence of a ‘coupling-uncoupling’ transition involving the protein hydrophilic section and the lipid polar regions, which may be triggered by the lipid bilayer pretransition. Our calculation of the average number of gauche bonds per lipid chain as a function of temperature and distance away from an isolated polypeptide or integral protein shows the extent of statically disordered lipid around such molecules. The range of this disorder depends upon temperature, particularly near the main transition.