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Biomedical subjects

M P Sheetz

Publications and source records attributed to M P Sheetz.

At least 19 recordsLinked to original sources
PubMed2005

Mechanisms of disease: the biophysical interpretation of the ECM affects physiological and pathophysiological cellular behavior.

Physiological and pathophysiological migration during the development and systemic spread of tumor cells requires a highly regulated interaction with the extracellular matrix. Sensing of the physical properties of the matrix as well as of forces exerted by the cell or acting on a cell is a prerequisite for productive migration. This review focuses on current concepts of the transmission of a physical stimulus into a biochemical signal in non-neuronal cells. Moreover, we summarize the current concepts on the regulation of affinity-modulation and regulation of protein-turnover for the formation and functionality of adhesion sites with special emphasis on the role of oncogenic signal transduction pathways such as Src family kinases and focal adhesion kinase.

Animals↗
PubMed2003

Pyk2 regulates multiple signaling events crucial for macrophage morphology and migration.

The biological role of the protein tyrosine kinase, Pyk2, was explored by targeting the Pyk2 gene by homologous recombination. Pyk2-/- mice are viable and fertile, without overt impairment in development or behavior. However, the morphology and behavior of Pyk2-/- macrophages were impaired. Macrophages isolated from mutant mice failed to become polarized, to undergo membrane ruffling, and to migrate in response to chemokine stimulation. Moreover, the contractile activity in the lamellipodia of Pyk2-/- macrophages was impaired, as revealed by measuring the rearward movement toward the nucleus of fibronectin-coated beads on the lamellipodia in opposition to an immobilizing force generated by optical tweezers. Consistently, the infiltration of macrophages into a carageenan-induced inflammatory region was strongly inhibited in Pyk2-/- mice. In addition, chemokine stimulation of inositol (1, 4, 5) triphosphate production and Ca2+ release, as well as integrin-induced activation of Rho and phosphatidyl inositol 3 kinase, were compromised in Pyk2-/- macrophages. These experiments reveal a role for Pyk2 in cell signaling in macrophages essential for cell migration and function.

Actins↗
PubMed2001

Binding of cross-linked glycosylphosphatidylinositol-anchored proteins to discrete actin-associated sites and cholesterol-dependent domains.

The mechanism by which cross-linked glycosylphosphatidylinositol (GPI)-anchored proteins are immobilized has been a mystery because both the binding to a transmembrane protein and attachment to a rigid cytoskeleton are needed. Using laser tweezers surface scanning resistance (SSR) technology, we obtained physical evidence for cross-linked GPI-anchored protein, Qa-2, binding to a transmembrane protein and for diffusion to discrete cytoskeleton attachment sites. At low levels of cross-linking of Qa-2 molecules, the resistance to lateral movement was that expected of monomeric lipid-anchored proteins, and no specific binding to cytoskeleton-attached structures was observed. When aggregates of the GPI-anchored protein, Qa-2, were scanned across plasma membranes, the background resistance was much higher than expected for a GPI-anchored protein alone and submicron domains of even higher resistance were observed (designated as elastic or non-elastic barriers) at a density of 82 (61 elastic and 21 small non-elastic barriers) per 100 microm(2). Elastic barriers involved weak but specific bonds to the actin cytoskeleton (broken by forces of 2 or 4 pN and were removed by cytochalasin D). Small non-elastic barriers (50-100 nm) depended upon membrane cholesterol and were closely correlated with caveolae density. Thus, cross-linked GPI-anchored proteins can diffuse through the membrane in complex with a transmembrane protein and bind weakly to discrete cytoskeleton attachment sites either associated with flexible actin networks or sphingolipid-cholesterol rich microdomains in live cell membranes. Our SSR measurements provide the first description of the physical characteristics of the interactions between rafts and stable membrane structures.

Actins↗
PubMed2001

Cell control by membrane-cytoskeleton adhesion.

The rates of mechanochemical processes, such as endocytosis, membrane extension and membrane resealing after cell wounding, are known to be controlled biochemically, through interaction with regulatory proteins. Here, I propose that these rates are also controlled physically, through an apparently continuous adhesion between plasma membrane lipids and cytoskeletal proteins.

Cell Adhesion↗
PubMed2001

Phospholipase C activation by anesthetics decreases membrane-cytoskeleton adhesion.

Many different amphiphilic compounds cause an increase in the fluid-phase endocytosis rates of cells in parallel with a decrease in membrane-cytoskeleton adhesion. These compounds, however, do not share a common chemical structure, which leaves the mechanism and even site of action unknown. One possible mechanism of action is through an alteration of inositol lipid metabolism by modifying the cytoplasmic surface of the plasma membrane bilayer. By comparing permeable amphiphilic amines used as local anesthetics with their impermeable analogs, we find that access to the cytoplasmic surface is necessary to increase endocytosis rate and decrease membrane-cytoskeleton adhesion. In parallel, we find that the level of phosphatidylinositol 4,5-bisphosphate (PIP(2)) in the plasma membrane is decreased and cytoplasmic Ca(2+) is increased only by permeable amines. The time course of both the decrease in plasma membrane PIP(2) and the rise in Ca(2+) parallels the decrease in cytoskeleton-membrane adhesion. Inositol labeling shows that phosphatidylinositol-4-phosphate levels are increased by the permeable anesthetics, indicating that lipid turnover is increased. Consistent with previous observations, phospholipase C (PLC) inhibitors block anesthetic effects on the PIP(2) and cytoplasmic Ca(2+) levels, as well as the drop in adhesion. Therefore, we suggest that PLC activity is increased by amine anesthetics at the cytoplasmic surface of the plasma membrane, which results in a decrease in membrane-cytoskeleton adhesion.

3T3 Cells↗
PubMed2000

Pilus retraction powers bacterial twitching motility.

Twitching and social gliding motility allow many gram negative bacteria to crawl along surfaces, and are implicated in a wide range of biological functions. Type IV pili (Tfp) are required for twitching and social gliding, but the mechanism by which these filaments promote motility has remained enigmatic. Here we use laser tweezers to show that Tfp forcefully retract. Neisseria gonorrhoeae cells that produce Tfp actively crawl on a glass surface and form adherent microcolonies. When laser tweezers are used to place and hold cells near a microcolony, retractile forces pull the cells toward the microcolony. In quantitative experiments, the Tfp of immobilized bacteria bind to latex beads and retract, pulling beads from the tweezers at forces that can exceed 80 pN. Episodes of retraction terminate with release or breakage of the Tfp tether. Both motility and retraction mediated by Tfp occur at about 1 microm s(-1) and require protein synthesis and function of the PilT protein. Our experiments establish that Tfp filaments retract, generate substantial force and directly mediate cell movement.

Fimbriae, Bacterial↗
PubMed2000

Characterization of myosin V binding to brain vesicles.

Myosin II and V are important for the generation and segregation of subcellular compartments. We observed that vesicular myosin II and V were associated with the protein scaffolding of a common subset of vesicles by density sedimentation, electron microscopy, and immunofluorescence. Solubilization of either myosin II or V was caused by polyphosphates with the following efficacy at 10 mM: for myosin II ATP-Mg(2+) = ATP = AMP-PNP (5'-adenylyl imidodiphosphate) > pyrophosphate = tripolyphosphate > tetrapolyphosphate = ADP > cAMP = Mg(2+); and for myosin V pyrophosphate = tripolyphosphate > ATP-Mg(2+) = ATP = AMP-PNP > ADP = tetrapolyphosphate > cAMP = Mg(2+). Consequently, we suggest solubilization was not an effect of phosphorylation, hydrolysis, or disassociation of myosin from actin filaments. Scatchard analysis of myosin V binding to stripped dense vesicles showed saturable binding with a K(m) of 10 nM. Analysis of native vesicles indicates that these sites are fully occupied. Together, these data show there are over 100 myosin Vs/vesicle (100-nm radius). We propose that polyphosphate anions bind to myosin II and V and induce a conformational change that disrupts binding to a receptor.

Adenosine Triphosphate↗
PubMed2000

Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion.

Binding interactions between the plasma membrane and the cytoskeleton define cell functions such as cell shape, formation of cell processes, cell movement, and endocytosis. Here we use optical tweezers tether force measurements and show that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) acts as a second messenger that regulates the adhesion energy between the cytoskeleton and the plasma membrane. Receptor stimuli that hydrolyze PIP2 lowered adhesion energy, a process that could be mimicked by expressing PH domains that sequester PIP2 or by targeting a 5'-PIP2-phosphatase to the plasma membrane to selectively lower plasma membrane PIP2 concentration. Our study suggests that plasma membrane PIP2 controls dynamic membrane functions and cell shape by locally increasing and decreasing the adhesion between the actin-based cortical cytoskeleton and the plasma membrane.

3T3 Cells↗
PubMed2000

Position-dependent linkages of fibronectin- integrin-cytoskeleton.

Position-dependent cycling of integrin interactions with both the cytoskeleton and extracellular matrix (ECM) is essential for cell spreading, migration, and wound healing. Whether there are regional changes in integrin concentration, ligand affinity or cytoskeleton crosslinking of liganded integrins has been unclear. Here, we directly demonstrate a position-dependent binding and release cycle of fibronectin-integrin-cytoskeleton interactions with preferential binding at the front of motile 3T3 fibroblasts and release at the endoplasm-ectoplasm boundary. Polystyrene beads coated with low concentrations of an integrin-binding fragment of fibronectin (fibronectin type III domains 7-10) were 3-4 times more likely to bind to integrins when placed within 0.5 microns vs. 0.5-3 microns from the leading edge. Integrins were not concentrated at the leading edge, nor did anti-integrin antibody-coated beads bind preferentially at the leading edge. However, diffusing liganded integrins attached to the cytoskeleton preferentially at the leading edge. Cytochalasin inhibited edge binding, which suggested that cytoskeleton binding to the integrins could alter the avidity for ligand beads. Further, at the ectoplasm-endoplasm boundary, the velocity of bead movement decreased, diffusive motion increased, and approximately one-third of the beads were released into the medium. We suggest that cytoskeleton linkage of liganded integrins stabilizes integrin-ECM bonds at the front whereas release of cytoskeleton-integrin links weakens integrin-ECM bonds at the back of lamellipodia.

3T3 Cells↗
PubMed2000

Cell spreading and lamellipodial extension rate is regulated by membrane tension.

Cell spreading and motility require the extension of the plasma membrane in association with the assembly of actin. In vitro, extension must overcome resistance from tension within the plasma membrane. We report here that the addition of either amphiphilic compounds or fluorescent lipids that expanded the plasma membrane increased the rate of cell spreading and lamellipodial extension, stimulated new lamellipodial extensions, and caused a decrease in the apparent membrane tension. Further, in PDGF-stimulated motility, the increase in the lamellipodial extension rate was associated with a decrease in the apparent membrane tension and decreased membrane-cytoskeleton adhesion through phosphatidylinositol diphosphate hydrolysis. Conversely, when membrane tension was increased by osmotically swelling cells, the extension rate decreased. Therefore, we suggest that the lamellipodial extension process can be activated by a physical signal (perhaps secondarily), and the rate of extension is directly dependent upon the tension in the plasma membrane. Quantitative analysis shows that the lamellipodial extension rate is inversely correlated with the apparent membrane tension. These studies describe a physical chemical mechanism involving changes in membrane-cytoskeleton adhesion through phosphatidylinositol 4,5-biphosphate-protein interactions for modulating and stimulating the biochemical processes that power lamellipodial extension.

3T3 Cells↗
PubMed2000

Outer membrane monolayer domains from two-dimensional surface scanning resistance measurements.

Cellular plasma membranes have domains that are defined, in most cases, by cytoskeletal elements. The outer half of the bilayer may also contain domains that organize glycosylphosphatidylinositol (GPI)-linked proteins. To define outer membrane barriers, we measured the resistive force on membrane bound beads as they were scanned across the plasma membrane of HEPA-OVA cells with optical laser tweezers. Beads were bound by antibodies to fluorescein-phosphatidylethanolamine (Fl-PE) or to the class I major histocompatibility complex (MHC class I) Qa-2 (a GPI-anchored protein). Two-dimensional scans of resistive force showed both occasional, resistive barriers and a velocity-dependent, continuous resistance. At the lowest antibody concentration, which gave specific binding, the continuous friction coefficient of Qa-2 was consistent with that observed by single-particle tracking (SPT) of small gold particles. At high antibody concentrations, the friction coefficient was significantly higher but decreased with increasing temperature, addition of deoxycholic acid, or treatment with heparinase I. Barriers to lateral movement (>3 times the continuous resistance) were consistently observed. Elastic barriers (with elastic constants from 1 to 20 pN/microm and sensitive to cytochalasin D) and small nonelastic barriers (<100 nm) were specifically observed with beads bound to the GPI-linked Qa-2. We suggest that GPI-linked proteins interact with transmembrane proteins when aggregated by antibody-coated beads and the transmembrane proteins encounter cytoplasmic barriers to lateral movement. The barriers to lateral movement are dynamic, discontinuous, and low in density.

Biological Transport↗
PubMed2000

The C-terminus of tubulin increases cytoplasmic dynein and kinesin processivity.

In motor movement on microtubules, the anionic C-terminal of tubulin has been implicated as a significant factor. Our digital analyses of movements of cytoplasmic dynein- and kinesin-coated beads on microtubules have revealed dramatic changes when the C-terminal region (2-4-kDa fragment) of tubulin was cleaved by limited subtilisin digestion of assembled microtubules. For both motors, bead binding to microtubules was decreased threefold, bead run length was decreased over fourfold, and there was a dramatic 20-fold decrease in diffusional movements of cytoplasmic dynein beads on microtubules (even with low motor concentrations where the level of bead motile activity was linear with motor concentration). The velocity of active bead movements on microtubules was unchanged for cytoplasmic dynein and slightly decreased for kinesin. There was also a decrease in the frequency of bead movements without a change in velocity when the ionic strength was raised. However, with high ionic strength there was not a decrease in run length or any selective inhibition of the diffusional movement. The C-terminal region of tubulin increased motor run length (processivity) by inhibiting "detachment" but without affecting velocity. Because the major motor binding sites of microtubules are not on the C-terminal tail of tubulin (), we suggest that the changes are the result of the compromise of a weakly attached state that is the lowest affinity step in both motors' ATPase cycles and is not rate limiting.

Animals↗
PubMed1999

Keratocytes pull with similar forces on their dorsal and ventral surfaces.

As cells move forward, they pull rearward against extracellular matrices (ECMs), exerting traction forces. However, no rearward forces have been seen in the fish keratocyte. To address this discrepancy, we have measured the propulsive forces generated by the keratocyte lamella on both the ventral and the dorsal surfaces. On the ventral surface, a micromachined device revealed that traction forces were small and rearward directed under the lamella, changed direction in front of the nucleus, and became larger under the cell body. On the dorsal surface of the lamella, an optical gradient trap measured rearward forces generated against fibronectin-coated beads. The retrograde force exerted by the cell on the bead increased in the thickened region of the lamella where myosin condensation has been observed (Svitkina, T.M., A.B. Verkhovsky, K.M. McQuade, and G. G. Borisy. 1997. J. Cell Biol. 139:397-415). Similar forces were generated on both the ventral (0.2 nN/microm(2)) and the dorsal (0.4 nN/microm(2)) surfaces of the lamella, suggesting that dorsal matrix contacts are as effectively linked to the force-generating cytoskeleton as ventral contacts. The correlation between the level of traction force and the density of myosin suggests a model for keratocyte movement in which myosin condensation in the perinuclear region generates rearward forces in the lamella and forward forces in the cell rear.

Actins↗
PubMed1999

Membrane expansion increases endocytosis rate during mitosis.

Mitosis in mammalian cells is accompanied by a dramatic inhibition of endocytosis. We have found that the addition of amphyphilic compounds to metaphase cells increases the endocytosis rate even to interphase levels. Detergents and solvents all increased endocytosis rate, and the extent of increase was in direct proportion to the concentration added. Although the compounds could produce a variety of different effects, we have found a strong correlation with a physical alteration in the membrane tension as measured by the laser tweezers. Plasma membrane tethers formed by latex beads pull back on the beads with a force that was related to the in-plane bilayer tension and membrane- cytoskeletal adhesion. We found that as cells enter mitosis, the membrane tension rises as the endocytosis rate decreases; and as cells exited mitosis, the endocytosis rate increased as the membrane tension decreased. The addition of amphyphilic compounds decreased membrane tension and increased the endocytosis rate. With the detergent, deoxycholate, the endocytosis rate was restored to interphase levels when the membrane tension was restored to interphase levels. Although biochemical factors are clearly involved in the alterations in mitosis, we suggest that endocytosis is blocked primarily by the increase in apparent plasma membrane tension. Higher tensions inhibit both the binding of the endocytic complex to the membrane and mechanical deformation of the membrane during invagination. We suggest that membrane tension is an important regulator of the endocytosis rate and alteration of tension is sufficient to modify endocytosis rates during mitosis. Further, we postulate that the rise in membrane tension causes cell rounding and the inhibition of motility, characteristic of mitosis.

3T3 Cells↗
PubMed1999

Myosin I contributes to the generation of resting cortical tension.

The amoeboid myosin I's are required for cellular cortical functions such as pseudopod formation and macropinocytosis, as demonstrated by the finding that Dictyostelium cells overexpressing or lacking one or more of these actin-based motors are defective in these processes. Defects in these processes are concomitant with changes in the actin-filled cortex of various Dictyostelium myosin I mutants. Given that the amoeboid myosin I's possess both actin- and membrane-binding domains, the mutant phenotypes could be due to alterations in the generation and/or regulation of cell cortical tension. This has been directly tested by analyzing mutant Dictyostelium that either lacks or overexpresses various myosin I's, using micropipette aspiration techniques. Dictyostelium cells lacking only one myosin I have normal levels of cortical tension. However, myosin I double mutants have significantly reduced (50%) cortical tension, and those that mildly overexpress an amoeboid myosin I exhibit increased cortical tension. Treatment of either type of mutant with the lectin concanavalin A (ConA) that cross-links surface receptors results in significant increases in cortical tension, suggesting that the contractile activity of these myosin I's is not controlled by this stimulus. These results demonstrate that myosin I's work cooperatively to contribute substantially to the generation of resting cortical tension that is required for efficient cell migration and macropinocytosis.

Animals↗
PubMed1999

Characteristics of a membrane reservoir buffering membrane tension.

When membrane-attached beads are pulled vertically by a laser tweezers, a membrane tube of constant diameter (tether) is formed. We found that the force on the bead (tether force) did not depend on tether length over a wide range of tether lengths, which indicates that a previously unidentified reservoir of membrane and not stretch of the plasma membrane provides the tether membrane. Plots of tether force vs. tether length have an initial phase, an elongation phase, and an exponential phase. During the major elongation phase, tether force is constant, buffered by the "membrane reservoir." Finally, there is an abrupt exponential rise in force that brings the tether out of the trap, indicating depletion of the membrane reservoir. In chick embryo fibroblasts and 3T3 fibroblasts, the maximum tether lengths that can be pulled at a velocity of 4 microm/s are 5.1 +/- 0. 3 and 5.0 +/- 0.2 microm, respectively. To examine the importance of the actin cytoskeleton, we treated cells with cytochalasin B or D and found that the tether lengths increased dramatically to 13.8 +/- 0.8 and 12.0 +/- 0.7 microm, respectively. Similarly, treatment of the cells with colchicine and nocodazole results in more than a twofold increase in tether length. We found that elevation of membrane tension (through osmotic pressure, a long-term elevation of tether force, or a number of transitory increases) increased reservoir size over the whole cell. Using a tracking system to hold tether force on the bead constant near its maximal length in the exponential phase, the rate of elongation of the tethers was measured as a function of tether force (membrane tension). The rate of elongation of tethers was linearly dependent on the tether force and reflected an increase in size of the reservoir. Increases in the reservoir caused by tension increases on one side of the cell caused increases in reservoir size on the other side of the cell. Thus, we suggest that cells maintain a plasma membrane reservoir to buffer against changes in membrane tension and that the reservoir is increased with membrane tension or disruption of the cytoskeleton.

3T3 Cells↗
PubMed1999

Membrane tether formation from blebbing cells.

Membrane tension has been proposed to be important in regulating cell functions such as endocytosis and cell motility. The apparent membrane tension has been calculated from tether forces measured with laser tweezers. Both membrane-cytoskeleton adhesion and membrane tension contribute to the tether force. Separation of the plasma membrane from the cytoskeleton occurs in membrane blebs, which could remove the membrane-cytoskeleton adhesion term. In renal epithelial cells, tether forces are significantly lower on blebs than on membranes that are supported by cytoskeleton. Furthermore, the tether forces are equal on apical and basolateral blebs. In contrast, tether forces from membranes supported by the cytoskeleton are greater in apical than in basolateral regions, which is consistent with the greater apparent cytoskeletal density in the apical region. We suggest that the tether force on blebs primarily contains only the membrane tension term and that the membrane tension may be uniform over the cell surface. Additional support for this hypothesis comes from observations of melanoma cells that spontaneously bleb. In melanoma cells, tether forces on blebs are proportional to the radius of the bleb, and as large blebs form, there are spikes in the tether force in other cell regions. We suggest that an internal osmotic pressure inflates the blebs, and the pressure calculated from the Law of Laplace is similar to independent measurements of intracellular pressures. When the membrane tension term is subtracted from the apparent membrane tension over the cytoskeleton, the membrane-cytoskeleton adhesion term can be estimated. In both cell systems, membrane-cytoskeleton adhesion was the major factor in generating the tether force.

Adhesiveness↗