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M P Sheetz

Publications and source records attributed to M P Sheetz.

At least 127 records · Page 7Linked to original sources
PubMed1988

The microtubule-dependent formation of a tubulovesicular network with characteristics of the ER from cultured cell extracts.

The formation of a dynamic tubulovesicular membrane network that resembles the endoplasmic reticulum (ER) has been observed in extracts of cultured chick embryo fibroblasts (CEF cells) using video-enhanced differential interference contrast microscopy. Initially, membranes in the CEF extracts appeared amorphous and aggregated, but with time, membrane tubules moved out along stationary microtubules. The membrane tubules formed new branches on intersecting microtubules and fused with other branches to form a network of interconnected polygons. The tubulovesicular network was solubilized by detergent and took on a beaded morphology in a hypotonic buffer. Formation of the tubulovesicular network required ATP and microtubules. The network did not contain elements of the plasma membrane, Golgi apparatus, or mitochondria but could be labeled with ER markers. We suggest that the tubulovesicular network contains components from the ER and is formed by membrane associated motors moving upon microtubules in a process we call microtubule-dependent tethering.

Adenosine Triphosphate↗
PubMed1988

Tracking kinesin-driven movements with nanometre-scale precision.

Several enzyme complexes drive cellular movements by coupling free energy-liberating chemical reactions to the production of mechanical work. A key goal in the study of these systems is to characterize at the molecular level mechanical events associated with individual reaction steps in the catalytic cycles of single enzyme molecules. Ideally, one would like to measure movements driven by single (or a few) enzyme molecules with sufficient temporal resolution and spatial precision that these events can be directly observed. Kinesin, a force-generating ATPase involved in microtubule-based intracellular organelle transport, will drive the unidirectional movement of microscopic plastic beads along microtubules in vitro. Under certain conditions, a few (less than or equal to 10) kinesin molecules may be sufficient to drive either bead movement or organelle transport. Here we describe a method for determining precise positional information from light-microscope images. The method is applied to measure kinesin-driven bead movements in vitro with a precision of 1-2 nm. Our measurements reveal basic mechanical features of kinesin-driven movements along the microtubule lattice, and place significant constraints on possible molecular mechanisms of movement.

Adenosine Triphosphate↗
PubMed1988

Nanometer-scale measurements using video light microscopy.

Video and digital image processing have been used to amplify the contrast of light microscopic images, making it possible to observe in real time the diffraction images of cell structures 10 times smaller than the Raleigh resolution limit of 0.2 micron. In this paper we discuss how quantitative analysis of diffraction images can be used to extract information about motion or structure at the nanometer level. This issue is considered in the context of a method for tracking the motion of kinesin-coated beads on microtubules with 1-2 nm precision (Gelles et al.:Nature 331:450-453, 1988).

Cell Movement↗
PubMed1988

Cultured cell extracts support organelle movement on microtubules in vitro.

Directed movements of organelles have been observed in a variety of cultured cells. To study the regulation and molecular basis of intracellular organelle motility, we have prepared extracts from cultured chick embryo fibroblasts (CEF cells) which support the movement of membraneous organelles along microtubules. The velocity, frequency and characteristics of organelle movements in vitro were similar to those within intact cells. Organelles and extract-coated anionic beads moved predominantly (80%) toward the minus ends of microtubules that had been regrown from centrosomes, corresponding to retrograde translocation. Similar microtubule-dependent organelle movements were observed in extracts prepared from other cultured cells (African green monkey kidney and 3T3 cells). Organelle motility was ATP and microtubule dependent. The frequency of organelle movement was inhibited by acidic (pH less than 7) or alkaline (pH greater than 8) solutions, high ionic strength ([ KCl] = 0.1 M), and the chelation of free magnesium ions. Treatment of the extracts with adenylyl imidodiphosphate (AMP-PNP, 7 mM), sodium orthovanadate (vanadate; Na3VO4, 20 microM), or N-ethylmaleimide (NEM, 2 mM) blocked all organelle motility. The decoration of microtubules with organelles was observed in the presence of AMP-PNP or vanadate. Motility was not affected by cytochalasin D (2 microM) or cAMP (1 mM). Kinesin (Mr = 116,000), an anterograde microtubule-based motor, was partially purified from the CEF extract by microtubule affinity purification in the presence of AMP-PNP, and was able to drive the movement of microtubule on glass coverslips. A similar preparation made in the presence of vanadate contained a different subset of proteins and did not support motility. These results demonstrate that intracellular organelle motility can be reproduced in vitro and provide the basis for investigating the roles of individual molecular components involved in the organelle motor complex.

Adenosine Triphosphate↗
PubMed1988

The role of kinesin and other soluble factors in organelle movement along microtubules.

Kinesin is a force-generating ATPase that drives the sliding movement of microtubules on glass coverslips and the movement of plastic beads along microtubules. Although kinesin is suspected to participate in microtubule-based organelle transport, the exact role it plays in this process is unclear. To address this question, we have developed a quantitative assay that allows us to determine the ability of soluble factors to promote organelle movement. Salt-washed organelles from squid axoplasm exhibited a nearly undetectable level of movement on purified microtubules. Their frequency of movement could be increased greater than 20-fold by the addition of a high speed axoplasmic supernatant. Immunoadsorption of kinesin from this supernatant decreased the frequency of organelle movement by more than 70%; organelle movements in both directions were markedly reduced. Surprisingly, antibody purified kinesin did not promote organelle movement either by itself or when it was added back to the kinesin-depleted supernatant. This result suggested that other soluble factors necessary for organelle movement were removed along with kinesin during immunoadsorption of the supernatant. A high level of organelle motor activity was recovered in a high salt eluate of the immunoadsorbent that contained only little kinesin. On the basis of these results we propose that organelle movement on microtubules involves other soluble axoplasmic factors in addition to kinesin.

Animals↗
PubMed1987

Characterization of the microtubule movement produced by sea urchin egg kinesin.

We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.

Adenosine Triphosphate↗
PubMed1987

Movements of vesicles on microtubules.

Many cytoplasmic vesicles are observed to move along microtubules. Often, bidirectional movement of particles is observed on a single microtubule. We have isolated one cytoplasmic motor, kinesin, and defined another, the axoplasmic retrograde factor, which are capable of powering anionic latex beads toward the plus and minus ends of microtubules, respectively. Observations of vesicle movements show that vesicles have a defined direction of movement and that vesicles copurify with a kinesin motor activity. Current evidence suggests the hypothesis that kinesin and the retrograde motors power vesicle movements in vivo by attachment to the appropriate vesicle.

Animals↗
PubMed1986

Vesicle movements and microtubule-based motors.

The movements of many cytoplasmic vesicles follow the paths of microtubules, some moving in one direction and others moving in the opposite direction on the same microtubule. Recently we have isolated one cytoplasmic motor, kinesin, and defined another, the axoplasmic retrograde factor, both of which are capable of powering anionic latex beads in both directions along polar microtubule arrays. Evidence summarized here supports but does not prove the hypothesis that kinesin and the retrograde motors are indeed responsible for powering vesicle movements.

Animals↗
PubMed1985

Monomeric Acanthamoeba myosins I support movement in vitro.

Acanthamoeba myosins IA and IB were found to have molecular weights of 159,000 and 150,000 and Stokes radii of 6.2 and 5.9 nm, respectively. Both enzymes have frictional ratios of 1.7. Myosin IA consists of 22% alpha-helix, 32% beta-structure, and 46% unordered structure, while myosin IB is 16% alpha-helix, 46% beta-structure, and 38% unordered. Both myosins remain monomolecular under conditions in which other myosins form filaments. Beads coated with myosin IA or IB move unidirectionally on actin cables of Nitella. Movement requires ATP and phosphorylation of the myosin I heavy chain which is also required for actin-activated Mg2+-ATPase activity. Movement is inhibited by myosin I antiserum that inhibits actin-activated ATPase activity. These studies establish that these nonfilamentous, monomolecular myosins with single heavy chains of 130,000 and 125,000 daltons (IA and IB, respectively) can support actin-dependent movement analogous to that supported by filamentous myosins.

Adenosine Triphosphatases↗
PubMed1985

Movement of organelles along filaments dissociated from the axoplasm of the squid giant axon.

Cytoplasmic filaments, separated from the axoplasm of the squid giant axon and visualized by video-enhanced differential interference contrast microscopy, support the directed movement of organelles in the presence of ATP. All organelles, regardless of size, move continuously along isolated transport filaments at 2.2 +/- 0.2 micron/sec. In the intact axoplasm, however, movements of the larger organelles are slow and saltatory. These movements may reflect a resistance to movement imposed by the intact axoplasm. The uniform rate of all organelles along isolated transport filaments suggests that a single type of molecular motor powers fast axonal transport. Organelles can attach to and move along more than one filament at a time, suggesting that organelles have multiple binding sites for this motor.

Adenosine Triphosphate↗
PubMed1985

Single microtubules from squid axoplasm support bidirectional movement of organelles.

Single filaments, dissociated from the extruded axoplasm of the squid giant axon and visualized by video-enhanced differential interference contrast microscopy, transport organelles bidirectionally. Organelles moving in the same or opposite directions along the same filament can pass each other without colliding, indicating that each transport filament has several tracks for organelle movement. In order to characterize transport filaments, organelle movements were first examined by video microscopy, and then the same filaments were examined by electron microscopy after rapid-freezing, freeze-drying, and rotary-shadowing. Transport filaments that supported bidirectional movement of organelles are 22 nm to 27 nm in diameter and have a substructure indicative of a single microtubule. Immunofluorescence showed that virtually all transport filaments contain tubulin. These results show that single microtubules can serve as a substratum for organelle movement, and suggest that an interaction between organelles and microtubules is the basis of fast axonal transport.

Animals↗
PubMed1985

Organelle, bead, and microtubule translocations promoted by soluble factors from the squid giant axon.

A reconstituted system for examining directed organelle movements along purified microtubules has been developed. Axoplasm from the squid giant axon was separated into soluble supernatant and organelle-enriched fractions. Movement of axoplasmic organelles along MAP-free microtubules occurred consistently only after addition of axoplasmic supernatant and ATP. The velocity of such organelle movement (1.6 micron/sec) was the same as in dissociated axoplasm. The axoplasmic supernatant also supported movement of microtubules along a glass surface and movement of carboxylated latex beads along microtubules at 0.5 micron/sec. The direction of microtubule movement on glass was opposite to that of organelle and bead movement on microtubules. The factors supporting movements of microtubules, beads, and organelles were sensitive to heat, trypsin, AMP-PNP and 100 microM vanadate. All of these movements may be driven by a single, soluble ATPase that binds reversibly to organelles, beads, or glass and generates a translocating force on a microtubule.

Adenosine Triphosphate↗
PubMed1985

Different axoplasmic proteins generate movement in opposite directions along microtubules in vitro.

Single microtubules from squid axoplasm support bidirectional movement of organelles. We previously purified a microtubule translocator (kinesin) that moves latex beads in only one direction along microtubules. In this study, a polar array of microtubules assembled off of centrosomes in vitro was used to demonstrate that kinesin moves latex beads from the minus to the plus ends of microtubules, a direction that corresponds to anterograde transport in the axon. A crude solubilized fraction from squid axoplasm (S1a), however, generates bidirectional movement of beads along microtubules. Retrograde bead movement (1.4 micron/sec) is inhibited by N-ethylmaleimide and 20 microM vanadate while anterograde movement (0.6 micron/sec) is unaffected by these agents. Furthermore, a monoclonal antibody against kinesin, when coupled to Sepharose, removes the anterograde, but not the retrograde, bead translocator from S1a. These results indicate that there is a retrograde bead translocator which is pharmacologically and immunologically distinct from kinesin.

Animals↗
PubMed1985

Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility.

Axoplasm from the squid giant axon contains a soluble protein translocator that induces movement of microtubules on glass, latex beads on microtubules, and axoplasmic organelles on microtubules. We now report the partial purification of a protein from squid giant axons and optic lobes that induces these microtubule-based movements and show that there is a homologous protein in bovine brain. The purification of the translocator protein depended primarily on its unusual property of forming a high affinity complex with microtubules in the presence of a nonhydrolyzable ATP analog, adenylyl imidodiphosphate. The protein, once released from microtubules with ATP, migrates on gel filtration columns with an apparent molecular weight of 600 kilodaltons and contains 110-120 and 60-70 kilodalton polypeptides. This protein is distinct in molecular weight and enzymatic behavior from myosin or dynein, which suggests that it belongs to a novel class of force-generating molecules, for which we propose the name kinesin.

Adenosine Triphosphate↗