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M P Sheetz

Publications and source records attributed to M P Sheetz.

At least 145 records · Page 8Linked to original sources
PubMed1985

Site-specific inhibition of myosin-mediated motility in vitro by monoclonal antibodies.

Monoclonal antibodies directed against seven different sites on Dictyostelium myosin (Peltz, G., J. A. Spudich, and P. Parham, 1985, J. Cell Biol., 100: 1016-1023) were tested for their ability to inhibit movement of myosin in vitro, using the Nitella-based myosin-mediated bead movement assay (Sheetz, M. P., R. Chasan, and J. A. Spudich, 1984, J. Cell Biol., 99: 1867-1871). To complement this functional assay, we located the binding sites of these antibodies by electron microscopy, using the rotary shadowing technique. One antibody bound to the 18,000-dalton light chain and inhibited movement completely. All of the remaining antibodies bound to various positions along the rod portion of the myosin molecule, which is approximately 1,800 A long. Antibodies that bound to the rod about 470, 680, and 1400 A from the head-tail junction did not alter myosin movement. One antibody appeared to bind very close to the head-tail junction and to inhibit movement 50%. Surprisingly, three antibodies that bound about 1,200 A from the head-tail junction inhibited movement completely. This inhibition did not depend on using intact IgG, since Fab' fragments had the same effect.

Antibodies, Monoclonal↗
PubMed1985

Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments.

In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.

Actins↗
PubMed1984

Movement of scallop myosin on Nitella actin filaments: regulation by calcium.

In order to determine if Ca2+ regulates scallop myosin movement on actin, we have measured motility of scallop myosin along actin filaments using a direct visual assay. This procedure consists of covalently linking myosin to 1-micron beads and pipetting them onto a parallel array of actin filaments located on the cytoplasmic face of a Nitella internodal cell. In the absence of Ca2+, scallop myosin-coated beads exhibit no directed motion; however, in the presence of pCa2+ of greater than 5.84, these beads undergo linear translocations with average velocities of 2.0 micron/s. This Ca2+ -sensitive motility requires the presence of regulatory light chains on the scallop myosin. Removal of regulatory light chains with 10 mM EDTA produces a "desensitized" myosin, no longer sensitive to Ca2+, which moves at rates of 0.09-0.3 micron in the presence or absence of Ca2+. Readdition of regulatory light chains to preparations of desensitized myosin once again confers Ca2+-sensitive motility. The Ca2+ dependence of scallop-myosin motility shows a sharp transition, consistent with the Ca2+ activation sensitivity of the actin-activated ATPase. Furthermore, relative rates of movement of calcium-regulated myosins from various molluscan species are consistent with their respective rates of ATP hydrolysis. Thus, myosin motility along actin filaments provides a sensitive and direct assay of myosin activity and is suitable for studying myosin regulation.

Actins↗
PubMed1984

ATP-dependent movement of myosin in vitro: characterization of a quantitative assay.

Sheetz and Spudich (1983, Nature (Lond.), 303:31-35) showed that ATP-dependent movement of myosin along actin filaments can be measured in vitro using myosin-coated beads and oriented actin cables from Nitella. To establish this in vitro movement as a quantitative assay and to understand better the basis for the movement, we have defined the factors that affect the myosin-bead velocity. Beads coated with skeletal muscle myosin move at a rate of 2-6 micron/s, depending on the myosin preparation. This velocity is independent of myosin concentration on the bead surface for concentrations above a critical value (approximately 20 micrograms myosin/2.5 X 10(9) beads of 1 micron in diameter). Movement is optimal between pH 6.8 and 7.5, at KCl concentrations less than 70 mM, at ATP concentrations greater than 0.1 mM, and at Mg2+ concentrations between 2 and 6 mM. From the temperature dependence of bead velocity, we calculate activation energies of 90 kJ/mol below 22 degrees C and 40 kJ/mol above 22 degrees C. Different myosin species move at their own characteristic velocities, and these velocities are proportional to their actin-activated ATPase activities. Further, the velocities of beads coated with smooth or skeletal muscle myosin correlate well with the known in vivo rates of myosin movement along actin filaments in these muscles. This in vitro assay, therefore, provides a rapid, reproducible method for quantitating the ATP-dependent movement of myosin molecules on actin.

Actins↗
PubMed1984

Polyphosphoinositides as regulators of membrane skeletal stability.

The membrane skeleton is a two-dimensional complex of actin, spectrinlike, and associated proteins which lies on the cytoplasmic face of most plasma membranes. Components of this complex are believed to control the lateral mobility of integral membrane proteins as well as influence cell shape and motility. In earlier studies we observed that the addition of polyphosphorylated inositol lipids could increased membrane skeleton dissociation. In preliminary studies of leukocyte function we have observed that increased chemotaxis with Trental addition is correlated with increased polyphosphoinositide levels. Consequently, we suggest that polyphosphoinositides contribute to, if not are requisite for, cellular mobility.

Animals↗
PubMed1984

Glucose-6-phosphate-dehydrogenase-deficient erythrocytes have an impaired shape recovery mechanism.

In the human erythrocyte, the maintenance of the biconcave disc shape is important for cell viability as well as cell function. Previous studies have indicated the involvement of the hexose monophosphate shunt in the recovery of discoid shape after perturbation of echinocytic agents. In glucose-6-phosphate-dehydrogenase-deficient (Gd-) erythrocytes, the shunt activity is significantly decreased; thus, it might be expected that the shape recovery rate of Gd- erythrocytes would be decreased. We show here that shape recovery rates in the presence of the shunt stimulator methylene blue are as much as fivefold lower in Gd- erythrocytes. We also show that the protease inhibitor, N-alpha-tosyl-1-phenylalanine-chloromethyl ketone, has no effect on shape recovery in Gd-, whereas it increases normal cell shape recovery rates by 10-30-fold at 50 microM and causes cupping at 200 microM (see companion article by Alhanaty et al.). These changes are not due to reticulocytosis, as other hemolytic disorders do not show such changes. Further, both chronic hemolyzing Gd and A Gd variants show similar abnormal shape recovery behavior, whereas the extent of hemolysis is quite different between variants. Thus, the activity of the hexose monophosphate shunt appears to have a dramatic effect on the rate of reversal of echinocytosis. The lack of shunt activity of Gd cells would necessarily impair their ability to recover normal shape after perturbation.

2,4-Dinitrophenol↗
PubMed1984

Cell membrane shape control--effects of chloromethyl ketone peptides.

The shape of the human erythrocyte is normally maintained in vivo as a biconcave disc for 120 days. In vitro, the cell shape can be altered readily by amphipathic compounds; however, given time and an energy source, the cells can recover the discoid morphology. An active shape control mechanism is postulated to regulate erythrocyte shape. The shape recovery process is a necessary element in reversing perturbations of shape and is basic to our understanding of how membrane shape is altered. We report here that the process of shape recovery from crenation is dramatically accelerated upon pretreatment of the cells with micromolar (20-100 microM) concentrations of chloromethyl ketone peptides [such as N-alpha-tosyl-L-phenylalanine-chloromethyl ketone (tos-pheCH2Cl)]. Such pretreatments do not appear to affect cellular viability, as judged by their normal biconcave disc shape, their sensitivity to crenators, their lactic acid production, or the ATP-dependent shape change of the purified membranes. Treatment with high concentrations of tos-pheCH2Cl does cause normal cells to become stomatocytic by an energy-requiring process, i.e., it requires glucose, incubation at 37 degrees C, and will not occur in ATP-depleted cells. We suggest that the chloromethyl ketone peptides affect a metabolic process that is associated with the hexose monophosphate (HMP) shunt. Through the alteration of the HMP shunt metabolism, they modify an active stomatocytic process in the erythrocyte that can correct for the perturbation caused by crenators. Implications of these findings for analogous phenomena in cultured cells are discussed.

2,4-Dinitrophenol↗
PubMed1983

Movement of myosin-coated structures on actin cables.

Myosin-coated spheres from 0.6 to 120 microns in diameter move in vitro on a substratum of polar arrays of actin cables derived from the alga Nitella. The force for this movement is provided by skeletal muscle myosin since it is ATP-dependent, and N-ethylmaleimide (NEM) inactivation of the myosin blocks movement. These observations demonstrate that attachment of myosin in a random orientation to structures will enable those structures to move along polar arrays of actin filaments.

Actins↗
PubMed1983

A localized pattern photobleaching method for the concurrent analysis of rapid and slow diffusion processes.

A scanning pattern photobleaching method for the analysis of lateral transport is described and discussed. Fluorescence bleaching with a localized pattern allows for the concurrent analysis of motions over two very different characteristic distances: xi 0(-1), the repeat distance of the pattern, and W, the linear dimension of the illuminated region. The former motion is deduced from the decay of the modulation amplitude (of period xi 0(-1) of fluorescence scans with the attenuated pattern, the latter from the recovery of the average fluorescence intensity. Such analysis should prove useful for the study of samples with a wide range of diffusion coefficients, and for the separation of effects arising from lateral diffusion and association dynamics. Theoretical analyses are presented for three related problems: (a) the effect of pattern localization on the decay of the modulation amplitude, (b) the effect of the pattern modulation on the recovery of the average local fluorescence intensity, and (c) the effect of a limited diffusion space (with linear dimensions of only a few pattern periods) on the decay of the modulation amplitude.

Diffusion↗
PubMed1983

Bilayer sensor model of erythrocyte shape control.

On the basis of recent studies a model of erythrocyte shape control is presented. This model is based on the assumption that the erythrocyte shape is normally determined by relative surface tensions of the two membrane bilayer surfaces. Our studies have indicated that the erythrocyte shape is preserved and can be restored upon perturbation by an active process. Consequently, it is proposed that in the "bilayer sensor" model the cells "sense" changes in the relative surface tensions, activating a process that balances the tensions so as to create a biconcave disc shape (reaching a new steady state which is observed as long as the perturbant is present). Experimental evidence has indicated that there is a mechanism to sense shape perturbations and two mechanisms to change shape through either crenation or cupping.

Chlorpromazine↗
PubMed1983

Membrane skeletal dynamics: role in modulation of red cell deformability, mobility of transmembrane proteins, and shape.

The dynamics of interactions in the membrane skeleton appear to control a variety of critical red cell membrane properties. Particularly sensitive parameters are deformability under prolonged shear and glycoprotein lateral diffusion rates. Because the dynamics of skeletal interactions can be controlled by polyanionic metabolite levels and other factors, it is suggested that metabolic abnormalities can cause skeletal dysfunction as well as abnormalities involving skeletal protein mutations. The membrane skeleton appears to be secondary to the bilayer couple in determining erythrocyte shape. Skeleton structure and dynamics do appear to influence lipid asymmetry and, by inference, the surface properties of the membrane, which will affect shape. Recent findings have shown that the erythrocyte can control its shape, and that shape control is related to hexose monophosphate shunt pathway activity. Consequently, defects in metabolism of the HMP shunt as well as structural protein abnormalities could result in abnormal cell shapes. In conclusion, the dynamics of the membrane skeleton and associated protein interactions appear to be central to many normal red cell functions and abnormal functions in disease. Much more complete knowledge of the molecular bases of these correlations, however, is required to understand fully the roles of skeletal dynamics in red cell functions.

Adenosine Triphosphate↗
PubMed1981

Matrix control of protein diffusion in biological membranes.

Lateral diffusion coefficients of fluorescently labeled lipids and integral membrane proteins were determined in the membranes of normal and spectrin-deficient spherocytic mouse erythrocytes by the technique of fluorescence redistribution after photobleaching. The results were used to generate a mathematical description of a matrix-control model of membrane protein diffusion. In the spherocytic cells, which lack the principal components of the cytoskeletal matrix of normal cells, the diffusion coefficients of lipid (1.5 +/- 0.5 X 10(-8) cm2/s) and protein (2.5 +/- 0.6 X 10(-9) cm2/s) differ only by a factor of 6, close to the difference predicted on the basis of size by the two-dimensional bilayer continuum model of Saffman and Delbrück [Saffman, P. G. l& Delbrück, M. (1975) Proc. Natl. Acad. Sci. USA 72, 3111-3113]. In contrast, the membranes of normal cells show a lipid diffusion coefficient (1.4 +/- 0.5 X 10(-8) cm2/s) that is some 300-fold greater than that of the membrane proteins (4.5 +/- 0.8 X 10(-11) cm2/s). Analysis of these results, based on the hypothesis that protein diffusion in normal membranes is sterically hindered by a labile matrix, yields an effective matrix surface viscosity consistent with the viscoelastic mechanical properties of the membranes. Thus, a relationship is established between the deformation characteristics of the membrane and the lateral mobility of proteins suspended in the membrane.

Animals↗
PubMed1981

Control of the erythrocyte membrane shape: recovery from the effect of crenating agents.

Intact erythrocytes become immediately crenated upon addition of 2,4-dinitrophenol (DNP) or pyrenebutyric acid (PBA). However, when cells are incubated at 37 degrees C in the presence of the crenating agents with glucose, they gradually (4--8 h) recover the normal biconcave disc form. The recovery process does not reflect a gradual inactivation of DNP or PBA since fresh cells are equally crenated by the supernatant from the recovered cells. Further, after recovery and removal of the crenating agents, cells are found to be desensitized to the readdition of DNP as well as to the addition of PBA, but they are more sensitive to cupping by chlorpromazine. This alteration in the cell membrane responsiveness was reversible upon further incubation in the absence of DNP. Recovery is dependent upon cellular metabolic state since an energy source is needed and incubation with guanosine but not adenosine will accelerate conversion to the disc shape. It is suggested that the conversion of cells from crenated to disc shape in the presence of the crenators, represents an alteration or rearrangement of membrane components rather than a redistribution of the crenators within the membrane. This shape recovery process may be important for erythrocyte shape preservation as well as shape control in other cells.

Adenosine↗
PubMed1981

Phosphate metabolite regulation of spectrin interactions.

The deformability of the erythrocyte membrane is believed to depend upon component interactions in the spectrin, actin and band 4.1 complex. Phosphate metabolites, such as 2,3-diphosphoglycerate (2,3 DPG) will dissociate spectrin from actin and band 4.1. This dissociation by 2,3 DPG is highly pH dependent but does not involve divalent cations, 2,3 DPG hydrolysis or spectrin dephosphorylation. In intact erythrocytes the concentrations of 2,3 DPG and the lipid, triphosphatidyl inositol, are sufficient to cause increased labilization in the spectrin, actin and band 4.1 network.

2,3-Diphosphoglycerate↗
PubMed1980

2,3-Diphosphoglycerate and ATP dissociate erythrocyte membrane skeletons.

Since ATP and 2,3-diphosphoglycerate cause an increase in the lateral mobility of integral membrane proteins in the erythrocyte (Schindler, M., Koppel, D., and Sheetz, M. P. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 1457-1461), we have studied their effects on the membrane skeletal complex or shell (composed of spectrin, actin, and bands 4.1 (78,000 daltons) and 4.9 (50,000 daltons)) and its interaction with the erythrocyte membrane. Both phosphate compounds dissociated the delipidated shell complex, with half-maximal dissociation at 2.5 mM 2,3-diphosphoglycerate and 8 mM ATP, whereas equivalent concentrations of EDTA did not. Concomitant with complex dissociation, spectrin was solubilized but band 4.1 and actin remained in a complexed or polymeric form. When proteins which were involved in linking spectrin to the membrane were present on the shell, higher concentrations of the phosphate compounds still dissociated the complex but less spectrin was solubilized. Treatment of erythrocyte membranes with the same phosphate compounds caused membrane vesiculation but no proteins were solubilized. We suggest that ATP and 2,3-diphosphoglycerate, at concentrations which are normally present in erythrocytes, can weaken associations in the shell but will not dissociate the complex from membrane attachment sites.

Adenosine Triphosphate↗
PubMed1980

Lateral mobility of integral membrane proteins is increased in spherocytic erythrocytes.

Alterations of glycoprotein distribution and lateral mobility in cell membranes can provide transmembrane signals for several membrane-related phenomena. Control of the transmembranous events has been ascribed to interaction between submembranous protein matrices (or 'cytoskeletons') and membrane glycoproteins. A consequence of such interaction would be differential inhibition of protein lateral diffusion in biological membranes. Measurements of the lateral diffusion coefficients of membrane proteins, in fact, have generally yielded values much less than were predicted for unhindered diffusion in a fluid bilayer. The mouse spherocytic erythrocyte, which lacks the major components of the normal erythrocyte membrane matrix (composed of spectrin, actin, bands 4.1 and 4.9 (ref. 16), in the nomenclature of Fairbanks et al.), provides a unique system for a direct evaluation of the effect of the matrix on protein lateral mobility. After using a modification of the technique of fluorescence redistribution after photobleaching (FRAP), we report here that membrane proteins diffuse about 50 times faster in spherocytic than in normal mouse erythrocytes.

Animals↗