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M Schliwa

Publications and source records attributed to M Schliwa.

At least 73 records · Page 4Linked to original sources
PubMed1991

Nucleotide specificities of anterograde and retrograde organelle transport in Reticulomyxa are indistinguishable.

Membrane-bound organelles move bidirectionally along microtubules in the freshwater ameba, Reticulomyxa. We have examined the nucleotide requirements for transport in a lysed cell model and compared them with kinesin and dynein-driven motility in other systems. Both anterograde and retrograde transport in Reticulomyxa show features characteristic of dynein but not of kinesin-powered movements: organelle transport is reactivated only by ATP and no other nucleoside triphosphates; the Km and Vmax of the ATP-driven movements are similar to values obtained for dynein rather than kinesin-driven movement; and of 15 ATP analogues tested for their ability to promote organelle transport, only 4 of them did. This narrow specificity resembles that of dynein-mediated in vitro transport and is dissimilar to the broad specificity of the kinesin motor (Shimizu, T., K. Furusawa, S. Ohashi, Y. Y. Toyoshima, M. Okuno, F. Malik, and R. D. Vale. 1991. J. Cell Biol. 112: 1189-1197). Remarkably, anterograde and retrograde organelle transport cannot be distinguished at all with respect to nucleotide specificity, kinetics of movement, and the ability to use the ATP analogues. Since the "kinetic fingerprints" of the motors driving transport in opposite directions are indistinguishable, the same type of motor(s) may be involved in the two directions of movement.

Adenosine Triphosphatases↗
PubMed1991

Viscoelastic properties of vimentin compared with other filamentous biopolymer networks.

The cytoplasm of vertebrate cells contains three distinct filamentous biopolymers, the microtubules, microfilaments, and intermediate filaments. The basic structural elements of these three filaments are linear polymers of the proteins tubulin, actin, and vimentin or another related intermediate filament protein, respectively. The viscoelastic properties of cytoplasmic filaments are likely to be relevant to their biologic function, because their extreme length and rodlike structure dominate the rheologic behavior of cytoplasm, and changes in their structure may cause gel-sol transitions observed when cells are activated or begin to move. This paper describes parallel measurements of the viscoelasticity of tubulin, actin, and vimentin polymers. The rheologic differences among the three types of cytoplasmic polymers suggest possible specialized roles for the different classes of filaments in vivo. Actin forms networks of highest rigidity that fluidize at high strains, consistent with a role in cell motility in which stable protrusions can deform rapidly in response to controlled filament rupture. Vimentin networks, which have not previously been studied by rheologic methods, exhibit some unusual viscoelastic properties not shared by actin or tubulin. They are less rigid (have lower shear moduli) at low strain but harden at high strains and resist breakage, suggesting they maintain cell integrity. The differences between F-actin and vimentin are optimal for the formation of a composite material with a range of properties that cannot be achieved by either polymer alone. Microtubules are unlikely to contribute significantly to interphase cell rheology alone, but may help stabilize the other networks.

Actin Cytoskeleton↗
PubMed1990

Force generation of organelle transport measured in vivo by an infrared laser trap.

Organelle transport along microtubules is believed to be mediated by organelle-associated force-generating molecules. Two classes of microtubule-based organelle motors have been identified: kinesin and cytoplasmic dynein. To correlate the mechanochemical basis of force generation with the in vivo behaviour of organelles, it is important to quantify the force needed to propel an organelle along microtubules and to determine the force generated by a single motor molecule. Measurements of force generation are possible under selected conditions in vitro, but are much more difficult using intact or reactivated cells. Here we combine a useful model system for the study of organelle transport, the giant amoeba Reticulomyxa, with a novel technique for the non-invasive manipulation of and force application to subcellular components, which is based on a gradient-force optical trap, also referred to as 'optical tweezers'. We demonstrate the feasibility of using controlled manipulation of actively translocating organelles to measure direct force. We have determined the force driving a single organelle along microtubules, allowing us to estimate the force generated by a single motor to be 2.6 x 10(-7) dynes.

Amoeba↗
PubMed1989

Head and tail.

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Adenosine Triphosphatases↗
PubMed1988

An ATPase with properties expected for the organelle motor of the giant amoeba, Reticulomyxa.

The rapid, vectorial, microtubule-associated transport of organelles is believed to be mediated by specific mechanochemical transducers. Recent studies of various metazoan cells have allowed the identification of novel microtubule-dependent translocator molecules capable of promoting microtubule gliding across glass surfaces and translocation of inert beads along microtubules. These translocators could be involved in force generation for directional organelle movements in vivo. Here we report the identification of a microtubule-binding protein with characteristics expected for an organelle translocator in the giant freshwater amoeba Reticulomyxa. This factor has an apparent relative molecular mass (Mr) of 440,000 (440K) and sediments at 20-22S in sucrose-density gradients. It binds to microtubules under conditions of ATP depletion, possesses an ATPase activity and is sensitive to ultraviolet-induced, vanadate-dependent cleavage. Although its pharmacological properties differ from those of axonemal dynein, it can be considered to be a variant of cytoplasmic dynein. The Reticulomyxa high-molecular-weight protein (HMWP) promotes rapid, bidirectional movement of latex beads along Reticulomyxa microtubules in vitro at an average speed of 3.6 micron s-1. This protein, therefore, is a likely candidate for a microtubule-dependent motor.

Adenosine Triphosphatases↗
PubMed1988

Melanophore death and disappearance produces color metamorphosis in the polychromatic Midas cichlid (Cichlasoma citrinellum).

We describe the histological basis of color metamorphosis in the polychromatic Midas cichlid, Cichlasoma citrinellum. Eight percent of the individuals in a natural population transform from gray with black markings to orange, simultaneously losing their ability to adjust coloration in response to background and social context. This trait is inherited. Light- and electron microscopy revealed that this transformation is a two-step process. First, the melanophores die, then macrophage-like cells remove the debris. As a result of this initial process, the underlying xanthophores become visible, producing the orange coloration. A similar process may occur in individuals that further transform to white, or go directly from gray to white.

Animals↗
PubMed1987

Release of enzymes of intermediary metabolism from permeabilized cells: further evidence in support of a structural organization of the cytoplasmic matrix.

Cultured BSC-1 cells were exposed to the mild ionic detergent, Brij 58, and the time course of the release of three enzymes of intermediary metabolism (lactate dehydrogenase, aldolase, and creatine phosphokinase) was determined spectrophotometrically. Their release correlates well with the overall decrease in structural complexity of the cytoplasmic matrix. However, each of the three enzymes tested has its own characteristic time-dependent release profile, a result suggesting enzyme-specific variability in their association with the cytomatrix. Cells lysed for 5 min in Brij 58 and then transferred to detergent-free glycolysis medium were able to produce lactate from glucose, an observation consistent with the idea that all enzymes of the glycolytic pathway remained in the cytomatrix. These observations are consistent with a structural rather than viscous organization of the cytoplasm and suggest that cytoplasmic components other than cytoskeletal filaments are able to form parafilamentous, metastable complexes.

Animals↗
PubMed1986

Cytoskeletal architecture and motility in a giant freshwater amoeba, Reticulomyxa.

Reticulomyxa is a large, multinucleated freshwater protozoan with striking intracellular transport. Cytoplasmic streaming and saltatory movements of individual organelles (at rates of up to 25 micron/sec) are observed within the naked cell body and the extensive reticulate peripheral network of fine cytoplasmic strands. As demonstrated by video-enhanced light microscopy, individual organelles move only when associated with cytoskeletal linear elements. The linear elements are composed of mixed colinear bundles of microtubules and actin filaments, which form the backbone of the reticulopodial network. The constant branching, sprouting, and fusion of network strands suggest unique membrane properties and an unusually dynamic cytoskeleton. The electrophoretic mobility of Reticulomyxa tubulins and the lack of crossreactivity with several antibodies known to react with many plant and animal tubulins suggest that they may differ from other tubulins more widely than might be expected. Reticulomyxa's large size, the rapidity and pervasiveness of the two forms of transport, and the simple and ordered cytoskeleton make the organism well suited for future studies on the mechanisms of intracellular transport.

Actin Cytoskeleton↗
PubMed1986

Relationship between the organization of actin bundles and vinculin plaques.

The temporal pattern of the formation and dissolution of vinculin patches during experimental manipulation of the state of actin within the cell was studied. Cytochalasin D-induced retraction and disappearance of stress fibers is followed, with a brief delay, by the dissolution of vinculin-containing patches and the coordinated redistribution of both actin and vinculin into newly formed amorphous aggregates or foci. Recovery from cytochalasin treatment begins with a transformation of these foci into doughnut-shaped assemblies in which actin and vinculin are precisely co-localized. The emergence and growth of filament bundles is paralleled by the appearance of faint vinculin patches that gradually increase in size in parallel with the stress fibers. If stress fibers are stabilized by microinjected rhodamine-phalloidin against stimuli that normally induce a coordinated redistribution of actin and vinculin, also the vinculin patches persist. These observations indicate that treatments influencing the state of actin in the cell have corresponding effects on the stability of vinculin patches and suggest a strong interdependency of actin and vinculin organization.

Actins↗
PubMed1986

Motility of cultured fish epidermal cells in the presence and absence of direct current electric fields.

The motile behavior and cytoskeletal structures of fish epidermal cells (keratocytes) in the presence and absence of direct current (DC) electric fields were examined. These cells spontaneously show highly directional locomotion in culture, migrating at rates of up to 1 micron/s. When DC electric fields between 0.5 and 15 V/cm are applied, single epidermal cells as well as cell clusters and cell sheets migrate towards the cathode. Cell clusters and sheets break apart into single migratory cells in the upper range of these field strengths. Cell shape and morphology are unaltered when the keratocytes are guided by an electric field. Neither the spontaneous locomotion nor the electrically guided motility were found to be microtubule dependent. 1 mM La3+, 10 mM Co2+, 50 microM verapamil, and 50 microM nitrendipine (calcium channel antagonists) reversibly inhibited lamellipod formation and cell locomotion in both spontaneously migrating and electrically guided cells. Ciba-Geigy Product 28392, which stimulates the opening of calcium channels, and is a competitive inhibitor of nitrendipine, has no effect on the locomotion of keratocytes. Cell motility was also unaffected by hyperpolarizing and depolarizing (low and high K+) media. It is argued that while a tissue cell may accommodate changes in resting membrane potential without becoming more or less motile, the cell may not be able to counterbalance the effects of depolarization and hyperpolarization simultaneously. In this context, a gradient of membrane potential, which is induced by an external DC electric field, will serve as a persistent stimulus for cell locomotion.

Animals↗
PubMed1986

Reactivation of organelle movements along the cytoskeletal framework of a giant freshwater ameba.

The peripheral feeding network of the giant freshwater ameba Reticulomyxa can be easily and rapidly lysed to produce an extensive, stable, and completely exposed cytoskeletal framework of colinear microtubules and microfilaments. Most of the organelles that remain attached to this framework resume rapid saltatory movements at rates of up to 20 micron/s if ATP is added. This lysed model system is also capable of other forms of motility, namely an active splaying of microtubule bundles and bulk streaming. Reactivation does not occur with other nucleoside triphosphates, requires Mg ions, is insensitive to even high concentrations of erythro-9-(3-[2-hydroxynonyl]) adenine, is sensitive to vanadate only at concentrations of approximately 100 microM, and is inhibited by N-ethylmaleimide at concentrations greater than 100 microM. The physiology of this reactivation suggests an organelle transport motor distinct from cytoplasmic dynein and possibly the recently described kinesin. This system can serve as a model for elucidating the mechanisms of intracellular transport and, in addition, provides a unique opportunity to examine associations between microtubules and microfilaments.

Actin Cytoskeleton↗
PubMed1986

Reticulomyxa: a new model system of intracellular transport.

Reticulomyxa is a large multinucleated freshwater protozoan that provides a new model system in which to study intracellular transport and cytoskeletal dynamics. Within the cell body and reticulopodial network, rapid, visually striking saltatory organelle motility as well as bulk cytoplasmic streaming can be readily observed. In addition, the cytoskeletal elements within these strands undergo dynamic splaying and fusing rearrangements, which can be visualized by video-enhanced light microscopy. A reactivatable lysed cell model has been developed that appears to preserve, and therefore permits examination of, these three forms of motility in a more controlled environment. Individual organelle movements are microtubule-based and have similarities to, but also differences from, the recently described kinesin-based transport. This lysed cell model can be further manipulated to provide native, ordered, completely exposed networks of either microtubules or microfilaments, or a combination of both, and thus may serve as a versatile motility assay system in which to examine the movement of exogenously added isolated organelles or latex beads.

Actin Cytoskeleton↗
PubMed1986

Motility in the siphonous green alga Bryopsis. I. Spatial organization of the cytoskeleton and organelle movements.

In the giant marine green alga Bryopsis the chloroplasts are attached with their flattened, ventral sides to the inner surface of the cortical cytoplasm. They move at speeds up to 60 microns/min in the direction of the long axis of the cell either in a coordinated fashion or independently of each other. Intracellular sedimentation of chloroplasts by centrifugation leaves an intact cell cortex in which the movement of mitochondria and nuclei--normally obscured by chloroplasts--can be observed. Mitochondria display a saltatory type of movement alongside an extensive, two-dimensional system of phase-translucent channels. Nuclei appear to be entangled in the channel system and move in an unusual, rolling fashion. With a new technique involving the microsurgical removal of the chemically fixed cytoplasm from the confinement of the cell wall, this unique cell type is made accessible to immunocytochemical procedures. Microtubules (MT) can be visualized using a variety of tubulin antibodies, while actin only reacts with one monoclonal antibody out of several antibodies tested. Microtubules form a dense, two-dimensional palisade of bundles extending longitudinally in the cortical cytoplasm. Parallel arrays of actin fibers closely, but not exclusively, colocalize with the MT bundles. Particularly strong actin staining is observed near converging MT bundles underneath the tip regions of the chloroplasts. Because of the extensive superposition of actin and MTs, both cytoskeletal elements could potentially cooperate in creating the diversity of organelle movements in this alga. The respective roles of MTs and actin in chloroplast movement are experimentally tested in the accompanying paper.

Cell Nucleus↗