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M P Sheetz

Publications and source records attributed to M P Sheetz.

At least 73 records · Page 4Linked to original sources
PubMed1995

A microtubule-associated protein (MAP2) kinase restores microtubule motility in embryonic brain.

Motility driven by the microtubule motors, kinesin and cytoplasmic dynein, is inhibited by MAP2 (López, L. A., and Sheetz, M. P. (1993) Cell Motil. Cytoskeleton 24, 1-16). The MAP2 inhibition is reversed by a kinase that is co-purified with chicken embryonic MAP2, completely releasing MAP2 from the microtubules. We have identified this activity with a kinase, embryonic MAP2 kinase (M(r) = 100,000), which phosphorylates MAP2 at serine amino acid residues. This kinase is c-AMP independent and inhibited by potassium fluoride and glycerol 2-phosphate. Only the phosphorylation produced by embryonic MAP2 kinase can change the affinity of MAP2 by microtubules. Bovine MAP2 kinase, Cdc2 kinase, mitogenic activated protein kinase, and the NIMA kinase are able to phosphorylate MAP2 but do not change the affinity for microtubules. In vivo, embryonic MAP2 kinase could play a major role in the regulation of motility and positioning of membranous organelles within the cells even at substoichiometric levels.

Animals↗
PubMed1995

Kinectin, an essential anchor for kinesin-driven vesicle motility.

The membrane anchor for the molecular motor kinesin is a critical site involved in intracellular membrane trafficking. Monoclonal antibodies specific for the cytoplasmic surface of chick brain microsomes were used to define proteins involved in microtubule-dependent transport. One of four antibodies tested inhibited plus-end-directed vesicle motility by approximately 90 percent even as a monovalent Fab fragment and reduced kinesin binding to vesicles. This antibody bound to the cytoplasmic domain of kinectin, an integral membrane protein of the endoplasmic reticulum that binds to kinesin. Thus, kinectin acted as a membrane anchor protein for kinesin-driven vesicle motility.

Animals↗
PubMed1995

In vitro acrylamide exposure alters growth cone morphology.

Acrylamide intoxication leads to degeneration of the longest axons of the central and peripheral nervous systems in humans and laboratory animals. Axonal derangements resulting from in vivo acrylamide exposure are first noted within synapses of the longest axons before involving more proximally located axonal segments or shorter axons, thus illustrating the specificity of acrylamide for the terminal axonal regions. As a possible model system for investigating the mechanism of toxicity of acrylamide on the distal axon, we exposed neurite-extending chick dorsal root ganglion (DRG) cells to acrylamide in vitro and then examined growth cones for alterations in morphology and function. Exposing DRG explants to media containing from 0.125 to 1.0 mM acrylamide for 16 hr leads to specific and dose-responsive alterations of growth cone morphology including: a nearly total loss of filopodial elements, the preservation of highly active but two-dimensional lamellar structures, an inappropriate extension of the axonal cytoskeleton into the forward region of most growth cones, and a frequent breakdown of the central and peripheral growth cone domains. The sulfhydryl alkylating agents ethacrynic acid, iodoacetamide, and iodoacetic acid were tested and none produced acrylamide-like morphological alterations at any dose. DRG cultures were also exposed to the neurotoxic acrylamide analogs glycidamide, N-hydroxy-methacrylamide (HM-ACR), and methacrylamide (M-ACR). At concentrations of 0.25 to 1.0 mM, glycidamide exposure resulted in acrylamide-like growth cone alterations. HM-ACR exposure also resulted in growth cones that were acrylamide-like but only at concentrations > 1.5 mM. M-ACR did not produce acrylamide-like growth cones at doses of up to 16.6 mM. Thus, in vitro exposure of DRG explants to acrylamide and two neurotoxic acrylamide analogs leads to reproducible and specific morphological alterations that are dose-dependent and separable from the effects of sulfhydryl alkylation.

Acrylamide↗
PubMed1995

The effect of acrylamide and other sulfhydryl alkylators on the ability of dynein and kinesin to translocate microtubules in vitro.

Chronic exposure to acrylamide leads to a dying-back axonopathy afflicting the longest axons of all tested mammalian and avian species. Prior to the onset of acrylamide-induced axonal degeneration, alterations in axonal fast transport have been consistently reported to be more severe for the retrograde than the anterograde direction. The putative retrograde motor protein, dynein, is compromised by exposure to the sulfhydryl-alkylating agent N-ethylmaleimide (NEM) at concentrations far below those required to inactivate kinesin, the putative anterograde motor protein. Since acrylamide is capable of alkylating protein sulfhydryl moieties, we tested whether a direct exposure of purified kinesin or dynein to acrylamide would result in an impairment of either enzyme's ability to translocate microtubules. Motor activity was assayed by sequentially adsorbing either kinesin or dynein to acid-washed coverslips, treating with an alkylating agent or control solution, adding microtubules and ATP, and finally imaging and quantifying the binding and gliding of microtubules using video-enhanced differential interference contrast (VE-DIC) microscopy. In comparison to controls, incubation of dynein with NEM, ethacrynic acid, or iodoacetic acid resulted in dose-dependent decreases in the amount and rate of microtubule gliding, but increases in irreversible high-affinity microtubule binding. In contrast, exposure of dynein to 1-100 mM solutions of acrylamide did not significantly alter either the binding or gliding of microtubules (a molar/hour exposure to acrylamide equivalent to 50 times that which causes retrograde transport deficits in vivo). Likewise, kinesin motility parameters were not significantly affected by acrylamide concentrations up to 100 mM while NEM solutions > 100 microM led to significant losses in the ability of kinesin to bind MT. These data indicate that acrylamide does not significantly interact with bound (adsorbed) kinesin or dynein, implying that the mechanism by which acrylamide interferes with fast axonal transport in vivo is by interaction with other factor(s) that govern the movement of vesicles.

Acrylamide↗
PubMed1995

Single cytoplasmic dynein molecule movements: characterization and comparison with kinesin.

Cytoplasmic dynein is a major microtubule motor for minus-end directed movements including retrograde axonal transport. To better understand the mechanism by which cytoplasmic dynein converts ATP energy into motility, we have analyzed the nanometer-level displacements of latex beads coated with low numbers of cytoplasmic dynein molecules. Cytoplasmic dynein-coated beads exhibited greater lateral movements among microtubule protofilaments (ave. 5.1 times/microns of displacement) compared with kinesin (ave. 0.9 times/micron). In addition, dynein moved rearward up to 100 nm over several hundred milliseconds, often in correlation with off-axis movements from one protofilament to another. We suggest that single molecules of cytoplasmic dynein move the beads because 1) there is a linear dependence of bead motility on dynein/bead ratio, 2) the binding of beads to microtubules studied by laser tweezers is best fit by a first-order Poisson, and 3) the run length histogram of dynein beads follows a first-order decay. At the cellular level, the greater disorder of cytoplasmic dynein movements may facilitate transport by decreasing the duration of collisions between kinesin and cytoplasmic dynein-powered vesicles.

Adenosine Triphosphate↗
PubMed1995

Mechanical properties of neuronal growth cone membranes studied by tether formation with laser optical tweezers.

Many cell phenomena involve major morphological changes, particularly in mitosis and the process of cell migration. For cells or neuronal growth cones to migrate, they must extend the leading edge of the plasma membrane as a lamellipodium or filopodium. During extension of filopodia, membrane must move across the surface creating shear and flow. Intracellular biochemical processes driving extension must work against the membrane mechanical properties, but the forces required to extend growth cones have not been measured. In this paper, laser optical tweezers and a nanometer-level analysis system were used to measure the neuronal growth cone membrane mechanical properties through the extension of filopodia-like tethers with IgG-coated beads. Although the probability of a bead attaching to the membrane was constant irrespective of treatment; the probability of forming a tether with a constant force increased dramatically with cytochalasin B or D and dimethylsulfoxide (DMSO). These are treatments that alter the organization of the actin cytoskeleton. The force required to hold a tether at zero velocity (F0) was greater than forces generated by single molecular motors, kinesin and myosin; and F0 decreased with cytochalasin B or D and DMSO in correlation with the changes in the probability of tether formation. The force of the tether on the bead increased linearly with the velocity of tether elongation. From the dependency of tether force on velocity of tether formation, we calculated a parameter related to membrane viscosity, which decreased with cytochalasin B or D, ATP depletion, nocodazole, and DMSO. These results indicate that the actin cytoskeleton affects the membrane mechanical properties, including the force required for membrane extension and the viscoelastic behavior.

Adenosine Triphosphate↗
PubMed1995

Nuclear spreads: I. Visualization of bipartite ribosomal RNA domains.

Visualization of nuclear architecture is key to the understanding of the association between RNA synthesis and processing. This architecture is obscured by the high density of components in most nuclei. We have developed a method of spreading nuclei and nucleoli that reduces overlap of weakly associated components. Strong interactions among nuclear components are not disrupted by this method. Spread nucleoli remained structurally distinct and functionally competent in ribosomal RNA synthesis. Nascent ribosomal RNA colocalized with RNA polymerase I and fibrillarin, a protein required for processing of ribosomal RNA. Colocalization of nascent transcripts and fibrillarin was seen in nucleoli spread over several microns, suggesting a strong interaction. These data suggest that nucleoli are superassemblies of bipartite domains, each composed of a ribosomal RNA synthesis center tightly associated with areas likely to be involved in ribosomal RNA processing.

Animals↗
PubMed1995

Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis.

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.

Amino Acid Sequence↗
PubMed1995

Integrin-cytoskeletal interactions in neuronal growth cones.

Development of the nervous system requires that neuronal growth cones, in coordination with growing axons, migrate along precise paths defined by specific extracellular matrix cues until they encounter their targets. Laminin promotes growth cone migration through receptors such as the integrins, but the underlying physical mechanism is poorly understood. We have investigated the cytoskeletal associations and surface dynamics of endogenous beta 1 integrins in chick dorsal root ganglion growth cones migrating on laminin. A single-beam optical gradient trap was used to place 0.5-micron-diameter polystyrene beads conjugated with anti-beta 1 integrin monoclonal antibodies at desired locations on the growth cone surface. We found a substantial increase in the stable attachment of these beads, with subsequent slow rearward motion, on the front periphery of the growth cone compared to the base. The surface dynamics of smaller aggregates of integrin were explored by monitoring the temporal and spatial displacements of 40-nm-diameter gold particles coated with anti-beta 1 integrin antibodies. The small particles were transported preferentially to the growth cone periphery by brief directed excursions interspersed with periods of diffusion. In addition, the leading edge of the growth cone was supported to a greater extent by an actin-dependent cytoskeleton that resisted mechanical tether formation. Such a regional differentiation of the growth cone has not been documented previously and has implications for the mechanism of growth cone migration and guidance.

Animals↗
PubMed1995

Cargo-activated ATPase activity of kinesin.

We have measured the ATPase activity of squid optic lobe kinesin bound to polystyrene beads in the presence of microtubules. We find that there is a substantial increase (> 10-fold) in the microtubule-activated ATPase activity for bead-bound kinesin over free kinesin. We tentatively attribute such cargo-activated ATPase activity to the presence of a self-inhibited form of kinesin in solution, which becomes activated when bound to a bead in the presence of alpha-casein. Further experiments are underway to unravel this phenomenon and, in addition, to associate the traveling distance of beads with the observed ATPase rate to determine the average number of ATP consumed per kinesin-bead per micron of travel along microtubule.

Adenosine Triphosphatases↗
PubMed1994

Coordinate depression of bradykinin receptor recycling and microtubule-dependent transport by taxol.

Significant cardiovascular side effects have limited the use of taxol as an anticancer drug. A link between decreased plasma membrane dynamics and taxol has been implied because taxol can inhibit intracellular vesicle movements. Reduced membrane recycling caused by taxol could inhibit agonist-evoked Ca2+ signaling within endothelial cells, resulting in endothelium-dependent vasodilation. Bradykinin and ATP are two agonists that evoke Ca2+ transients in endothelial cells. Since the bradykinin receptor-agonist complex is internalized and recycled whereas the ATP agonist-receptor complex is not, we expected that a taxol inhibition of recycling would decrease bradykinin but not ATP receptor activity. We found that taxol depresses (i) the frequency (to 41% of control) and velocity (to 55% of control) of microtubule-dependent vesicle transport and (ii) bradykinin-evoked cytosolic Ca2+ transients (to 76% of control) in bovine aortic endothelial cells. In studying bradykinin receptor desensitization, which reflects receptor recycling, we demonstrate that taxol inhibits bradykinin-evoked Ca2+ transients by 50%. Taxol did not significantly alter ATP-evoked Ca2+ transients in either single-exposure or desensitization experiments. We suggest that taxol's reduction of bradykinin-evoked Ca2+ transients is due to altered microtubule-dependent membrane recycling. This report describes taxol's ability to alter plasma membrane composition through effects on vesicle transport and membrane trafficking pathways. This finding provides a possible mechanism by which taxol can substantially alter cardiovascular function.

Adenosine Triphosphate↗
PubMed1994

Regulation of kinesin activity by phosphorylation of kinesin-associated proteins.

The mechanochemical motor proteins of the kinesin and cytoplasmic dynein families play important roles in microtubule-based intracellular motility. Although movement and distribution of organelles like secretory granules, vesicles, endoplasmic reticulum, and chromosomes depend on the activity of these motor proteins, little is known about the regulation of this movement. We report here that the hyperphosphorylation of components of the kinesin complex by treatment with okadaic acid increases kinesin motor activity at least 2-fold. The stimulation was observed using both a granule motility assay and a microtubule gliding assay, indicating that phosphorylation enhances the activity of the motor itself, rather than the affinity of the motor for membrane organelles. Under stimulatory conditions, three proteins that co-purify with kinesin (with mobilities of 150, 79, and 73 kDa) are consistently hyperphosphorylated. Dephosphorylation of these proteins reduces kinesin activity to basal levels. Therefore, we conclude that kinesin motor activity is directly modulated by the phosphorylation state of kinesin-associated proteins.

Animals↗
PubMed1994

Truncation mutants define and locate cytoplasmic barriers to lateral mobility of membrane glycoproteins.

The lateral mobility of cell membrane glycoproteins is often restricted by dynamic barriers. These barriers have been detected by measurements of fluorescence photobleaching and recovery (FPR) and barrier-free path (BFP). To define the location and properties of the barriers, we compared the lateral mobility, measured by FPR and BFP, of wild-type class I major histocompatibility complex (MHC) membrane glycoproteins with the lateral mobility of mutant class I MHC glycoproteins truncated in their cytoplasmic domains. Mutants with 0 or 4 residues in the cytoplasmic domain were as mobile as lipid-anchored class I MHC molecules, molecules whose lateral mobility is relatively unrestricted by barriers. In contrast, mobility of class I MHC molecules with 7-residue cytoplasmic domains was as restricted as mobility of class I molecules with full-length, 31-residue cytoplasmic domains. Though some of the difference between the mobilities of mutants with 4- or 0-residue domains and the other class I molecules may be due to differences in the net charge of the cytoplasmic domain, FPR measurements of the mobility of molecules with 7-residue domains show that length of the cytoplasmic domain has an important influence on the lateral mobility. Model calculations suggest that the barriers to lateral mobility are 2-3 nm below the membrane bilayer.

Amino Acid Sequence↗
PubMed1994

Mechanics of myosin motor: force and step size.

How motor proteins induce mechanical movement at the molecular level has been a focus of biophysicists for a long time. While the whole picture is yet to be completely revealed, recent developments in looking at nanometer-scale movement with millisecond-time resolution driven by single motors have revealed important new details about the moving step size and amount of force generated per molecule.

Adenosine Triphosphate↗
PubMed1994

Cell migration by graded attachment to substrates and contraction.

The migration of fibroblastic cells in vitro involves the extension of lamellipodia, attachment of the cytoskeleton through the plasma membrane to the extracellular matrix, and the generation of force that pulls attachments rearward and the cell forward. Bulk flow of membrane or lipid relative to the cell outline cannot be detected; however, crosslinked glycoproteins attached to the cytoskeleton move rearward and diffusing particles are driven forward by a motor mechanism. The leading edge is the preferential site for the cytoskeleton to attach to crosslinked glycoproteins including integrins. Force for moving the cell forward can be generated either by a cortical contraction acting as a net to pull the endoplasm forward or by motors at the boundary of the endoplasm and ectoplasm pulling on the cortical actin. As the cortical actin is anchored to the external matrix more strongly at the front of the cell than at the rear, contraction will pull the cell forward. Such a model has important implications for the nature of the glycoprotein attachments to the cytoskeleton and the regional differences in membrane structure.

Animals↗
PubMed1993

Force of single kinesin molecules measured with optical tweezers.

Isometric forces generated by single molecules of the mechanochemical enzyme kinesin were measured with a laser-induced, single-beam optical gradient trap, also known as optical tweezers. For the microspheres used in this study, the optical tweezers was spring-like for a radius of 100 nanometers and had a maximum force region at a radius of approximately 150 nanometers. With the use of biotinylated microtubules and special streptavidin-coated latex microspheres as handles, microtubule translocation by single squid kinesin molecules was reversibly stalled. The stalled microtubules escaped optical trapping forces of 1.9 +/- 0.4 piconewtons. The ability to measure force parameters of single macromolecules now allows direct testing of molecular models for contractility.

Adenosine Triphosphate↗
PubMed1993

Steric inhibition of cytoplasmic dynein and kinesin motility by MAP2.

Using several in vitro motility assays, we found that motility driven by the microtubule (MT) motors, kinesin and cytoplasmic dynein, could be inhibited by MAP2 but not by tau protein or the MT-binding proteolytic fragment of MAP2. In MT gliding assays, even the presence of one MAP2 molecule per sixty-nine tubulin dimers caused an inhibition of about 75% of MT motility at low concentrations of both motors. The percent inhibition of motility decreased with increasing concentration of either motor, suggesting that the inhibition was the result of competition for access to the MT surface. The decrease in the number of moving MTs with MAP2 was correlated with an increase in the frequency of release of moving MTs from the motor-coated glass. In assays of in vitro vesicular organelle motility and formation of ER networks, the presence of MAP2 inhibited small vesicle movements and to a lesser extent ER network formation. To determine if competition for specific sites on the MT or coating of the MT surface inhibited motility, we used tau protein and the chymotryptic MT-binding fragments of MAP2 to coat MTs. No inhibition was observed and there was even an increase in the number of attached and moving MTs in the gliding assay with tau-coated MTs. Because MAP2, tau and the chymotryptic MT-binding fragments of MAP2 bind to the same domain on tubulin, masking of the MT surface sites does not appear responsible for the inhibition of motility by MAP2. Rather, we suggest that the sidearm of MAP2 interfered with the interaction of motors with MTs and caused a dramatic increase in the rate of MT release. In vivo, MAP2 could play a major role in the generation of cellular polarity even at substoichiometric levels by inhibiting transport on microtubules in specific domains of the cytoplasm.

Animals↗