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Biomedical subjects

M P Sheetz

Publications and source records attributed to M P Sheetz.

At least 55 records · Page 3Linked to original sources
PubMed1997

Phosphotransferases associated with the regulation of kinesin motor activity.

Kinesin, a plus-end-directed microtubule motor protein, functions in concert with accessory factors that have been shown to regulate enzyme activity and may also provide cargo specificity. This report identifies teh 79-kDa kinesin-associated phosphoprotein as a phosphoisoform of kinesin light chain. Increased phosphorylation of this light chain isoform is sufficient to account for the increase in kinesin-mediated microtubule-gliding activity. Additionally, it was found that the degree of phosphorylation of this isoform is regulated by a 100-kDa kinase and 150-kDa type 1 phosphatase. Both the kinesin light chain kinase and phosphatase co-purify with the kinesin heavy chain, suggesting that kinesin exists in a large complex capable of self-regulation.

Animals↗
PubMed1997

A micromachined device provides a new bend on fibroblast traction forces.

We have measured the traction forces generated by fibroblasts using a novel micromachined device that is capable of determining the subcellular forces generated by individual adhesive contacts. The front of migrating fibroblasts produced intermittent rearward forces whereas the tail produced larger forward directed forces. None of the forces were steady; they all had periodic fluctuations. The transition between forward and rearward traction forces occurred at the nucleus, not at the rear of the cell or the border between the endoplasm and the ectoplasm. We propose that the coupling of lamella extensions to fluctuating rearward tractions in front of the nuclear region move the front of a fibroblast forward, while force-facilitated release of rear adhesive contacts and anterior-directed tractions allow the region behind the nucleus to advance.

Animals↗
PubMed1997

Ligation of L-selectin through conserved regions within the lectin domain activates signal transduction pathways and integrin function in human, mouse, and rat leukocytes.

L-selectin is a cell surface adhesion receptor that mediates leukocyte rolling along vascular endothelium at sites of inflammation and lymphocyte attachment to endothelial cells within peripheral lymphoid tissues. Since ligation of L-selectin through its ligand recognition region may mimic physiologic ligand binding, a new panel of mAbs that engaged a conserved ligand-binding region within the lectin domains of human, mouse, and rat L-selectin were generated using L-selectin-deficient mice. Indeed, appropriate ligation of L-selectin generated transmembrane signals that resulted in immediate intercellular adhesion following cell-cell contact of lymphocytes, neutrophils, and L-selectin cDNA-transfected cells. Ab binding to only some epitopes within the lectin domain of L-selectin induced adhesion, while mAb binding to numerous other epitopes or other domains of L-selectin had no effect. PPME (yeast polyphosphomonoester core polysaccharide), a complex carbohydrate that mimics the natural L-selectin ligand, also induced potent intercellular adhesion. The induction of intercellular adhesion required cellular energy, an intact cytoskeleton, and cytoplasmic kinase activity as well as the membrane proximal and cytoplasmic domains of L-selectin. Therefore, ligation of L-selectin through specific ligand-binding epitopes identified by the mAbs generated in this study can generate transmembrane signals. Signaling through L-selectin may enhance leukocyte-endothelial cell interactions by serving as an activation/priming step during rolling, thereby promoting subsequent firm cell-cell adhesion in the presence of inflammatory mediators.

Amino Acid Sequence↗
PubMed1997

Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages.

To move forward, migrating cells must generate traction forces through surface receptors bound to extracellular matrix molecules coupled to a rigid structure. We investigated whether cells sample and respond to the rigidity of the anchoring matrix. Movement of beads coated with fibronectin or an anti-integrin antibody was restrained with an optical trap on fibroblasts to mimic extracellular attachment sites of different resistance. Cells precisely sense the restraining force on fibronectin beads and respond by a localized, proportional strengthening of the cytoskeleton linkages, allowing stronger force to be exerted on the integrins. This strengthening was absent or transient with antibody beads, but restored with soluble fibronectin. Hence, ligand binding site occupancy was required. Finally, phenylarsine oxide inhibited strengthening of cytoskeletal linkages, indicating a role for dephosphorylation. Thus, the strength of integrin-cytoskeleton linkages is dependent on matrix rigidity and on its biochemical composition. Matrix rigidity may, therefore, serve as a guidance cue in a process of mechanotaxis.

3T3 Cells↗
PubMed1997

n-ethylmaleimide and ethacrynic acid inhibit kinesin binding to microtubules in a motility assay.

Treatment of proteins in vitro with sulfhydryl (SH)-reactive compounds has been used successfully to determine protein regions critical for normal function. To probe structure-function relationships in the microtubule (MT) motor kinesin, the motor was treated with two SH reactive compounds, n-ethylmaleimide and ethacrynic acid, and its function was assayed by motility and co-sedimentation techniques. In the motility assay, treatment of kinesin either before or after adsorption to the glass surfaces of a flow cell was found to inhibit the ability of coverslip-bound kinesin to bind to MTs. Inactivation of MT binding was slow, required high molar excess of the SH-reactive drug, and was very sensitive to temperature. Inhibition of MT binding occurred well after complete modification of kinesin light chain, but paralleled modification of the kinesin heavy chain. The results point to a model in which one critical cysteine per kinesin heavy chain is relatively inaccessible to solvent. Surprisingly, when the interaction between modified kinesin and MTs was examined by a co-sedimentation assay, kinesin retained the ability to bind MTs. These contrasting results may be due to conformational differences in the kinesin molecule that exist in the two assays.

Animals↗
PubMed1997

Inversely correlated cycles in speed and turning in an ameba: an oscillatory model of cell locomotion.

Previous biophysical models of ameboid crawling have described cell movement in terms of a persistent random walk. Speed and orientation were treated in the latter model as independent and temporally homogeneous stochastic processes. We show here that, at least in the case of Dictyostelium discoideum, both speed control and reorientation processes involve a deterministic, periodic component. We also show that the processes are synchronized and negatively correlated, as was suggested by earlier findings. That is, increased turning correlates with periods of slow movement. Therefore, previous models are inconsistent with the behavior of cells. Using a heuristic approach, we have developed a mathematical model that describes the statistical properties of the cell's velocity and movement of its centroid. Our observations and the model are consistent with the phenomenological description of ameboid motility as a cyclic process of pseudopod extension and retraction.

Amoeba↗
PubMed1997

The secretion-coupled endocytosis correlates with membrane tension changes in RBL 2H3 cells.

Stimulated secretion in endocrine cells and neuronal synapses causes a rise in endocytosis rates to recover the added membrane. The endocytic process involves the mechanical deformation of the membrane to produce an invagination. Studies of osmotic swelling effects on endocytosis indicate that the increased surface tension is tightly correlated to a significant decrease of endocytosis. When rat basophilic leukemia (RBL) cells are stimulated to secrete, there is a dramatic drop in the membrane tension and only small changes in membrane bending stiffness. Neither the shape change that normally accompanies secretion nor the binding of ligand without secretion causes a drop in tension. Further, tension decreases within 6 s, preceding shape change and measurable changes in endocytosis. After secretion stops, tension recovers. On the basis of these results we suggest that the physical parameter of membrane tension is a major regulator of endocytic rate in RBL cells. Low tensions would stimulate endocytosis and high tensions would stall the endocytic machinery.

Animals↗
PubMed1997

Force effects on biochemical kinetics.

Force is an important component in the proper functioning of tissues and cells. In processes ranging from the contraction of muscles to the alignment of chromosomes at the metaphase plate, forces must be adjusted to the proper levels by cells. At the molecular level, it is clear that the motor molecules and other enzymes must respond to changes in mechanical forces by altering enzymatic function. Recent technical advances, primarily the atomic force microscope and laser tweezers, enable us to measure forces at the single molecule level to test how force is transduced into a change in enzyme activity. A priori, four basic mechanisms of coupling enzyme rate and force are considered. The mechanisms extend from the cellular to the molecular level. For example, polymer assembly rates and cytoskeletal matrix concentration are potentially modified by force in ways that feed back on critical enzyme rates. In studies of the known mechanosensitive enzymes, myosin and other motors, the bacterial flagellar rotor, and the F0F1 ATPase, the molecular mechanisms used to transduce force changes into activity changes have not been clearly defined, although it is reasonable to speculate about the nature of these mechanisms from the atomic structures and nanometer measurements of movement.

Animals↗
PubMed1997

Loss of cytoskeletal support is not sufficient for anoxic plasma membrane disruption in renal cells.

The goal of this study was to determine whether anoxic membrane disruption is initiated by loss of cytoskeletal support in rabbit renal proximal tubules (PT). We specifically tested 1) whether cytoskeletal perturbation affects membrane integrity under normoxia, 2) whether cytoskeletal perturbation potentiates anoxic membrane damage, and 3) whether the membrane protection by glycine depends on cytoskeletal integrity. Cytoskeletal perturbation was achieved with 10 microM cytochalasin D (CD) because it selectively disturbs F-actin organization and has similar effects as anoxia on the cytoskeleton of PT. During normoxia, CD caused decreased basal F-actin content, microvillar breakdown, and membrane-cytoskeleton dissociation, as revealed by the use of laser tweezers. However, membrane integrity was not altered by CD, as monitored by lactate dehydrogenase release. CD pretreatment of PT did not potentiate anoxic membrane damage. Finally, plasma membrane protection by glycine during anoxia remained in CD-pretreated PT despite loss of cytoskeletal support. These results demonstrate that loss of cytoskeletal support is not sufficient for anoxic plasma membrane disruption.

Animals↗
PubMed1997

Overexpression of MAP4 inhibits organelle motility and trafficking in vivo.

We previously prepared cell lines that inducibly overexpress MAP4, a microtubule (MT)-associated protein widely expressed in non-neuronal cells. Overexpression of either the full-length MAP4 molecule or its MT-binding domain, MTB, stabilized MTs and retarded cell growth, suggesting that overexpressed MAP4 impacts on MT-dependent functions in vivo. To test this hypothesis, we examined MT-based vesicle movements in living cells, using high resolution DIC microscopy. Overexpression of either MAP4 or MTB yielded a dose-dependent reduction in the frequency of MT-dependent organelle movements, relative to control cells. At steady state, both MAP4- and MTB-overexpressing cells showed unusual distributions of transferrin, LDL, dextran, and Golgi elements, as compared to control cells. MAP4 preferentially inhibited receptor-dependent uptake and degradation of LDL, and repositioning of Golgi elements after disruption by the drug, brefeldin A. L-MOCK cells treated with Taxol to stabilize the MTs to an extent equivalent to MAP4 overexpression did not show similar inhibition of vesicle motility or organellar trafficking, suggesting that deficits in organelle movements in vivo represent a direct effect of the presence of MAP4 or MTB, rather than an indirect effect of the stabilization of MTs by overexpressed MAP constructs. Our results show that MAP4 has the capacity to affect transport along MTs in vivo; these findings suggest a potential mechanism by which MAP4 could contribute to polarization or morphogenesis of cells.

Biological Transport↗
PubMed1996

Ligand binding regulates the directed movement of beta1 integrins on fibroblasts.

To enable cells to crawl, adhesion receptors such as integrins must bind to extracellular molecules and simultaneously interact with force-generating components of the cytoskeleton. We show here that the binding of extracellular ligand in living cells induces the attachment of beta1 integrins to the retrograde-moving cytoskeleton. Unliganded integrins are not associated with the rearward-moving cytoskeleton: gold particles attached to beta1 integrin by a monoclonal antibody diffuse in the membrane. However, addition of soluble RGD peptide (single-letter amino-acid code) or the use of fibronectin-coated gold particles causes the attachment of integrins to the rearward-moving cytoskeleton. Deletion of the beta1 cytoplasmic tail blocks cytoskeletal attachment. The directed movement of integrins in response to ligand indicates that ligand binding is the critical step in regulating organized receptor movement on the cell surface and the migration of adherent cells.

3T3 Cells↗
PubMed1996

Kinectin distribution in chicken nervous system.

Kinectin, a major kinesin receptor on endoplasmic reticulum, was visualized with anti-kinectin monoclonal antibodies (mAbs) in the adult chicken nervous system in comparison with kinesin immunostaining. Anti-kinectin mAbs punctately stained cell bodies and proximal dendrites of motor neurons in spinal cords. Axons of motor neurons were not stained with anti-kinectin mAbs, but stained heavily with anti-kinesin mAbs. This suggest that the kinesin receptor responsible for kinesin-driven anterograde fast axonal transport is different from kinectin. Anti-kinectin mAbs strongly stained neuronal cell bodies in spinal ganglion, nuclei in brainstem, cerebellar nuclei, striatum and cerebral cortex. Small neurons in cerebellar cortex and optic lobe showed relatively weak reaction, suggesting that the amount of kinectin correlates with the size of neuronal cell bodies.

Animals↗
PubMed1996

Targeting of motor proteins.

Microtubules are responsible for chromosome segregation and the movement and reorganization of membranous organelles. Many aspects of microtubule-based motility can be attributed to the action of motor proteins, producing force directed toward either end of microtubules. How these proteins are targeted to the appropriate organellar sites within the cell, however, has remained a mystery. Recent work has begun to define the targeting mechanism for two well-studied motor proteins, kinesin and cytoplasmic dynein.

Animals↗
PubMed1996

Modulation of membrane dynamics and cell motility by membrane tension.

The plasma membrane of most cells is drawn tightly over the cytoskeleton of the cell, resulting in a significant tension being developed in the membrane. The tension in the membrane can be calculated from the force required to separate it from the cytoskeleton; and the force itself can be measured rapidly by using laser tweezers. Recent observations indicate that decreasing membrane tension stimulates endocytosis and increasing tension stimulates secretion. Thus, membrane tension provides a simple physical mechanism to control the area of the plasma membrane. Here, we speculate that tension is a global parameter that the cell uses to control physically plasma membrane dynamics, cell shape and cell motility.

Journal Article↗
PubMed1996

Deformation and flow of membrane into tethers extracted from neuronal growth cones.

Membrane tethers are extracted at constant velocity from neuronal growth cones using a force generated by a laser tweezers trap. A thermodynamic analysis shows that as the tether is extended, energy is stored in the tether as bending and adhesion energies and in the cell body as "nonlocal" bending. It is postulated that energy is dissipated by three viscous mechanisms including membrane flow, slip between the two monolayers that form the bilayer, and slip between membrane and cytoskeleton. The analysis predicts and the experiments show a linear relation between tether force and tether velocity. Calculations based on the analytical results and the experimental measurements of a tether radius of approximately 0.2 micron and a tether force at zero velocity of approximately 8 pN give a bending modulus for the tether of 2.7 x 10(-19) N.m and an extraordinarily small "apparent surface tension" in the growth cone of 0.003 mN/m, where the apparent surface tension is the sum of the far-field, in-plane tension and the energy of adhesion. Treatments with cytochalasin B and D, ethanol, and nocodazole affect the apparent surface tension but not bending. ATP depletion affects neither, whereas large concentrations of DMSO affect both. Under conditions of flow, data are presented to show that the dominant viscous mechanism comes from the slip that occurs when the membrane flows over the cytoskeleton. ATP depletion and the treatment with DMSO cause a dramatic drop in the effective viscosity. If it is postulated that the slip between membrane and cytoskeleton occurs in a film of water, then this water film has a mean thickness of only approximately 10 A.

Animals↗
PubMed1996

Microtubule motor complexes moving membranous organelles.

Cellular polarization depends upon the asymmetric transport of components within the cell, often along microtubule paths. Kinesin and cytoplasmic dynein are the most abundant microtubule motors powering the transport of membranous vesicles. Many additional proteins are needed to control motility and co-ordinate movements with other cellular activities. In this review we will summarize our understanding of the motors, motor associated proteins and their function within cells. We suggest a cyclic model for much of membrane trafficking in which the membrane anchor for the motors, kinectin, plays a central role. In such a model up-regulation of transport results in a faster cycling of kinectin between kinesin and cytoplasmic dynein-dependent transport.

Animals↗
PubMed1995

Axon membrane flows from the growth cone to the cell body.

During the growth of axons, the surface area of the neuron increases dramatically. Membrane addition as well as exchange could contribute to rapid membrane dynamics or flow. Using diffusing latex beads to monitor membrane flow, we find that axonal membrane flows rapidly (7 microns/min) from growth cone to cell body during axon growth and that flow is inhibited by brefeldin A. To power this flow, there is a membrane lesion gradient from growth cone to cell body that could draw the membrane over the axon at that rate. Further, when an artificial flow is induced to the center of the axon by use of laser tweezers, the primary source of the membrane is from the growth cone. We suggest that during neuron growth, there is excess membrane added at the growth cone in chick dorsal ganglia (DRGs) that undergoes edcocytosis at the cell body, thereby creating a flow that can rapidly alter the content of the axon membrane.

Animals↗