Nocodazole, vinblastine and taxol at low concentrations affect fibroblast locomotion and saltatory movements of organelles

Microtubules (MTs) are essential for the maintenance of asymmetric cell shape and motility of fibroblasts. MTs are considered to function as rails for organelle transport to the leading edge. We investigated the relationship between the motility of Vero fibroblasts and saltatory movements of particles in their lamella Fibroblasts extended their leading edges into the experimental wound at a rate of 20+/-11 microm/h. Intracellular particles in the front parts of the polarized fibroblasts moved saltatorily mainly along the long axis of the cells. MT depolymerization induced by the nocodazole at a high concentration (1.7 microM) resulted in the inhibition of both fibroblast motility and saltatory movements of the particles. Taxol (1 microM) inhibited the fibroblast locomotion but not the saltatory movements. The saltatory movement pattern was disorganized by taxol by decreasing the portion of longitudinal saltations and consequently by increasing the part of saltations perpendicular to the cell long axis. This effect may be explained by disorganization of the MT network resulting from the inhibition of dynamic instability. To further investigate the relationships between the MT dynamics instability, saltatory movements, and fibroblast locomotion, we treated fibroblasts with microtubule drugs at low concentration (nocodazole, 170 nM; vinblastine, 50 nM; and taxol, 50 nM). All these drugs induced rapid disorganization of the saltatory movements and decreased the rate of cell locomotion. Simultaneously, the amount of acetylated (stable) MTs increased. The treatment also induced reversible changes in the actin meshwork. We suggest that decrease in the fibroblast locomotion rate in the case of MT stabilization occurred because of the appearance of numerous free MTs. Saltations along free MTs are poorly organized and, as a result, the number of organelles reaching the fibroblast leading edge decreases.     

Functions of cytoplasmic fibers in intracellular movements in BHK-21 cells

After trypsinization and replating, BHK-21 cells spread and change shape from a rounded to a fibroblastic form. Time-lapse movies of spreading cells reveal that organelles are redistributed by saltatory movements from a juxtanuclear position into the expanding regions of cytoplasm. Bidirectional saltations are seen along the long axes of fully spread cells. As the spreading process progresses, the pattern of saltatory movements changes and the average speed of saltations increases from 1.7 MICROMETER/S during the early stages of spreading to 2.3 micrometer/s in fully spread cells. Correlative electron microscope studies indicate that the patterns of saltatory movements that lead to the redistribution of organelles during spreading are closely related to changes in the degree of assembly, organization, and distribution of microtubules and 10-nm filaments. Colchicine (10 microgram/ml of culture medium) reversibly disassembles the microtubule-10-nm filament complexes which form during cell spreading. This treatment results in the disappearance of microtubules and the appearance of a juxtanuclear accumulation of 10-nm filaments. These changes closely parallel an inhibition of saltatory movements. Within 30 min after the addition of the colchicine, pseudopod-like extensions form rapidly at the cell periphery, and adjacent organelles are seen to stream into them. The pseudopods contain extensive arrays of actinlike microfilament bundles which bind skeletal-muscle heavy meromyosin (HMM). Therefore, in the presence of colchicine, intracellular movements are altered from a normal saltatory pattern into a pattern reminiscent of the type of cytoplasmic streaming seen in amoeboid organisms. The streaming may reflect either the activity or the contractility of submembranous microfilament bundles. Streaming activity is not seen in cells containing well-organized microtubule-10-nm filament complexes.     

Co-Localization of Particle Transport with Microtubules in Cytoplasmic Exudates of the Siphonous Green Alga Bryopsis

Organelles in the cortical cytoplasm of the siphonous green alga Bryopsis display various types of motile activities. One of them, saltatory movement along axially oriented linear tracks is typical for mitochondria and other small particles. A method is described which allows in vitro observation of such movements in thin layers of cytoplasm extruded from the alga and attached to a poly-l -lysine coated glass surface. By comparing video recordings of motile activities with the position of cytoskeletal elements visualized by immunofluorescence in the same area of a cytoplasmic exudate, it can be shown that tracks along which particles have moved in vitro are identical with microtubules (MTs). Depolymerization of MTs in the cytoplasmic exudates by MT-specific inhibitors stops particle movement, whereas depolymerization of actin filaments with cytochalasin D disrupts actin bundles but has little effect on particle motility. These data are consistent with the model of MT guided particle transport.     

Quantitative Measurement ed Pollen Tube Growth and Particle Movement

The growth of pollen tube and the cytoplasmic particle movements in pollen tube of Aloe zebrina Haw. were recorded by micro-video and measured by computer image analysis. The saltatory growth of pollen tube was observed. The movement velocity, diameter and the rate of flux of forward particles towards pollen tube tip were greater than those of backward particles. The results indicate that the cytoplasmic particle movements may play a role in transporting “building blocks” for pollen tube growth.     

Alteration of the distribution of intermediate filaments in PtK1 cells by acrylamide. II: Effect on the organization of cytoplasmic organelles

The distribution and motility of cytoplasmic particles was examined in PtK1 cells in which intermediate filament networks had been disrupted by acrylamide. In these cells, particles (mitochondria and vesicles) accumulated near the cell center although saltatory movements continued. This left a broad sheet of agranular cytoplasm at the periphery of the cell. Particles were capable of movement into this sheet. Intermediate filaments were absent in the peripheral cytoplasm although microtubules remained in a normal configuration. Particles apparently move along the microtubules. These results indicate that particle movement along microtubules is not dependent upon the normal configuration of intermediate filaments. It is suggested that intermediate filaments are necessary for normal organelle distribution and serve as a matrix with which particles can associate to maintain position.     

Structural Aspects of Saltatory Particle Movement

A variety of cells possess particles which show movements statistically different from Brownian movements. They are characterized by discontinuous jumps of 2–30 µ at velocities of 0.5–5 µ/sec or more. A detailed analysis of these saltatory, jumplike movements makes it most likely that they are caused by transmission of force to the particles by a fiber system in the cell outside of the particle itself. Work with isolated droplets of cytoplasm from algae demonstrates a set of fibers involved in both cytoplasmic streaming and saltatory motion, suggesting that both phenomena are related to the same force-generating set of fibers. Analysis of a variety of systems in which streaming and/or saltatory movement occurs reveals two types of fiber systems spatially correlated with the movement, microtubules and 50 A microfilaments. The fibers in Nitella (alga) are of the microfilament type. In other systems (melanocyte processes, mitotic apparatus, nerve axons) microtubules occur. A suggestion is made, based on work on cilia, that a microtubule-microfilament complex may be present in those cases in which only microtubules appear to be present, with the microfilament closely associated with or buried in the microtubule wall. If so, then microfilaments, structurally similar to smooth muscle filaments, may be a force-generating element in a very wide variety of saltatory and streaming phenomena.     

Cytoplasmic streaming inParamecium

Summary Certain species ofParamecium demonstrate rotational cytoplasmic streaming, in which most cytoplasmic particles and organelles flow along permanent route, in a constant direction. By means of novel methods of immobilization, observation and recording, some dynamic properties of cytoplasmic streaming have been described. It was found that the velocity profiles of coaxial layers of cytoplasm have a (parabolic) paraboidal shape and the mean output of cytoplasm flow in different examined zones of streaming is constant. As the consequence of randomly distributed elementary propulsion units within the cytoplasm, particles, which serve as markers of movement, exhibit movements of a saltatory nature; this form of movement is seen inParamecium streaming only in cases of error due to polarization of the saltating particles. Interaction of actin filaments and myosin is likely to occur under specific conditions in microcompartments of cytoplasm where local solations are generated eventually leading to contractions which might propagate on gelated neighbouring areas. Places of elementary contractions are scattered. Therefore the motile effect appears as streaming. Rotational cytoplasmic streaming inParamecium may serve as a convenient model for the study of the dynamics and function of cytoplasmic motility.     

Responses of spiking local interneurones in the locust to proprioceptive signals from the femoral chordotonal organ

Summary The responses of spiking local interneurones of a ventral midline population in the metathoracic ganglion of the locust,Schistocerca gregaria, to controlled movements of a proprioceptor, the femoral chordotonal organ (FCO) in a hindleg, were revealed by intracellular recording. Afferents from the FCO which signal specific features of the movement or angle of the femoro-tibial joint, can make direct excitatory synapses with particular interneurones in this population (Burrows 1987a).Some interneurones in this population are excited only by flexion, some only by extension, but others by both flexion and extension movements of the femoro-tibial joint. Interneurones excited by one direction of movement may be either unaffected, or inhibited by the opposite movement. The balance between excitation and inhibition is determined by the range over which the movement occurs, and can increase the accuracy of a representation of a movement.The response of some interneurones has tonic components, so that the angle of the joint over a certain range is represented in the frequency of their spikes. Different interneurones respond within different ranges of femoro-tibial angles so that information about the position of the joint is fractionated amongst several members of the population. These interneurones respond to repetitive movements, similar to those used by the locust during walking, with bursts of spikes whose number and frequency are determined by the repetition rate and amplitude of the movement. A brief movement of the FCO may induce effects which persist for many seconds and outlast the changed pattern of afferent spikes. The sign of such an effect depends upon the preceding history of stimulation.Other interneurones respond only to movement so that their response is more phasic. The velocities to which they respond fall within the range of those generated by twitches of the flexor and extensor tibiae muscles and the movements of the tibia during locomotion. Some interneurones respond only to a specific range of velocities because they are inhibited by all other movements. Some interneurones respond to repetitive movements with reliable bursts of spikes, whilst in others the frequency of spikes may be raised but may contain no cyclical information. All, however, produce the largest number of spikes during the first cycle of a repetitive movement.Inputs from the FCO may sum either with excitation generated by direct inputs from exteroceptors or with inhibition produced by other local interneurones as a result of afferent signals.These spiking local interneurones are essential elements in the integration of local reflexes initiated by signals from the FCO. For example, one ensures that the levator tarsi motor neurone is reflexly inhibited when the FCO signals an extension movement. Exteroceptive inputs from the ventral tarsus suppress the spikes in this interneurone and would prevent expression of the reflex when the tarsus is in contact with ground.Abbreviation FCO femoral chordotonal organ     

Motility in Echinosphaerium nucleofilum. II. Cytoplasmic contractility and its molecular basis

Echinosphaerium nucleofilum exhibits at least three kinds of movement: locomotion by the bending and shortening of its many axopodia, feeding by means of food-cup pseudopodia formed from its cortical cytoplasm, and saltatory motion of cytoplasmic particles, especially in the cortex and axopodia. Since previously presented evidence indicated that the microtubular axoneme is not essential for particle motion, the cytoplasm was investigated for the possible existence of contractile behavior and for the possible presence of linear elements other than microtubules. Cytoplasm can be isolated in physiological media in which rigor, relaxation, and contraction can be induced, as in muscle, by manipulating the concentrations of calcium ions and magnesium-adenosine triphosphate. Contraction is initiated by calcium ions at concentrations above 2.4 times 10-minus 7 M. The rigor-to-relaxation transition occurs at subthreshold calcium concentrations on the addition of 10-minus 3 M ATP. Negatively stained preparations of isolated cytoplasm show two types of filaments: thin filaments identified as cytoplasmic actin by virtue of their binding heavy meromyosin from striated muscle in characteristic arrowhead arrays, and thicker filaments which do not strictly resemble myosin aggregates from muscle or amoeba but could conceivably by myosin aggregated in an unfamiliar form.     

Microinjection into Acanthamoeba castellanii of monoclonal antibodies to myosin-II slows but does not stop cell locomotion

To study the in vivo role of myosin-II in Acanthamoeba castellanii, motile cells were microinjected with monoclonal antibodies raised against the myosin-II heavy chain. All injected cells underwent a transient shock response. It was found that although injection of buffer alone or of an endogenous Acanthamoeba protein decreased the motility of injected cells from 7 microns/min to approximately 3 microns/min, injection of monoclonal antibodies specific for myosin-II decreased motility further to approximately 0.8 micron/min. This effect was seen whether or not the monoclonal antibody to myosin-II inhibited the actomyosin-II MgATPase activity in vitro. Levels of antibody far in excess of endogenous myosin-II concentrations could not completely block amoeboid movement. The morphology of moving antimyosin-II-injected cells was unusual, suggesting a greater defect in the ability to retract the trailing edge of the cell rather than to extend the leading edge. Endosomes frequently disappeared from injected cells, and although buffer-injected cells rapidly recovered visible endosomes (50% recovery at 5 min), endosomes were not seen in antimyosin-II-injected cells until, on the average, approximately 50 min after injection. Injection of a nonspecific antibody or of a nonspecific exogenous protein (ovalbumin) also decreased the mobility of the injected cells beyond that of buffer-injected cells (to approximately 1 micron/min). These cells tended to recover endosomes more rapidly (approximately 25 min) than cells injected with antimyosin-II monoclonal antibodies. The inability of antibodies to myosin-II to inhibit completely any of the movements studied suggests that although myosin-II probably plays a role in these motilities, the cell either routinely uses or can draw upon another cytoplasmic motor to maintain locomotion, organelle movement, contractile vacuole activity, and endocytosis.     

Kinesin-1 tail autoregulation and microtubule-binding regions function in saltatory transport but not ooplasmic streaming

The N-terminal head domain of kinesin heavy chain (Khc) is well known for generating force for transport along microtubules in cytoplasmic organization processes during metazoan development, but the functions of the C-terminal tail are not clear. To address this, we studied the effects of tail mutations on mitochondria transport, determinant mRNA localization and cytoplasmic streaming in Drosophila. Our results show that two biochemically defined elements of the tail - the ATP-independent microtubule-binding sequence and the IAK autoinhibitory motif - are essential for development and viability. Both elements have positive functions in the axonal transport of mitochondria and determinant mRNA localization in oocytes, processes that are accomplished by biased saltatory movement of individual cargoes. Surprisingly, there were no indications that the IAK autoinhibitory motif acts as a general downregulator of Kinesin-1 in those processes. Time-lapse imaging indicated that neither tail region is needed for fast cytoplasmic streaming in oocytes, which is a non-saltatory bulk transport process driven solely by Kinesin-1. Thus, the Khc tail is not constitutively required for Kinesin-1 activation, force transduction or linkage to cargo. It might instead be crucial for more subtle elements of motor control and coordination in the stop-and-go movements of biased saltatory transport.     

The difference between random movement and chemotaxis. Effects of antitubulins on neutrophil granulocyte locomotion

The antitubulins demecolcine and podophyllic acid ethylhydrazide (SPI) were used in experiments designed to elucidate the role of centriole-associated cytoplasmic microtubules in the locomotion of human neutrophil granulocytes (PMNs). The PMN locomotion was studied as chemotaxis and as the velocity of random movement. The PMN chemotaxis was inhibited by demecolcine (0.01 μg/ml) and SPI (0.1 μg/ml), i.e. concentrations below the reported threshold ones for mitotic arrest in metaphase. The velocity of single PMNs during random movement was only slightly reduced by treatment with 0.5 μg/ml of SPI. PMN locomotion was not appreciably inhibited by SPI, 0.5 μg/ml. The discrepancies mentioned suggest that centriole-associated microtubules are essential structures in the PMN direction-finding or PMN directional movement of chemotaxis but not in the mechanism of PMN locomotion. The present observations might, at least in part, explain the beneficial effects of antitubulins on acute gout.     

Microinjected fluorescent polystyrene beads exhibit saltatory motion in tissue culture cells

Microinjected 0.26-micron fluorescent, carboxylated microspheres were found to display classical saltatory motion in tissue culture cells. The movement of a given particle was characterized by a discontinuous velocity distribution and was unaffected by the activity of adjacent particles. The microspheres were translocated at velocities of up to 4.7 micron/s and sometimes exhibited path lengths greater than 20 micron for a single saltation . The number of beads injected into a cell could range from a few to over 500 with no effect on the cell's ability to transport them. Neither covalent cross-linking nor preincubation of the polystyrene beads with various proteins inhibited the saltatory motion of the injected particles. The motion of the injected beads in cultured cells was reversibly inhibited by the microtubule poison nocodazole, under conditions in which actin-rich, nitrobenzoxadiazol - phallacidin -staining structures remain intact. Whole-cell high voltage electron microscopy of microinjected cells that were known to be moving the fluorescent microspheres revealed that the beads were embedded in the cytoplasmic matrix and did not appear to be membrane bound. The enhanced detectability of the fluorescent particles over endogenous organelles and the ability to modify the surfaces of the beads before injection may enable more detailed studies on the mechanism of saltatory particle motion.     

Speed of locomotion of the soil amoebaNaegleria gruberi in media of different ionic compositions with special reference to interactions with the substratum

Summary The effect of electrolytes on the speed of locomotion of amoebae on glass coverslips was studied in a perfusion chamber. The intervening gap between the ventral surface of the cell and the glass substrate in the various solutions was also studied by means of reflexion interference microscopy. It was found that the presence of electrolyte increased the speed of movement,e.g., the amoebae moved almost four times faster in 10 mM KCl than in deionized water. The ventral surface of the amoebae was brought closer to the substrate when increasing amounts of electrolyte were present in the medium. This two fold effect was fully reversible by replacement of electrolyte with deionized water. Solutions of the non-electrolytes sucrose or urea did not affect the behaviour of the amoebae with respect to their speed of movement or cell-substrate separation distance compared with that observed in deionized water. Thus we have demonstrated a direct relationship between the speed of amoeboid cell locomotion and the closeness of the cell to the substrate.     

Microinjected Polystyrene Beads Move Along Astral Rays in Sand Dollar Eggs

Movements of polystyrene beads along astral rays of the sperm aster and the mitotic aster were investigated in eggs of the sand dollars, Clypeaster japonicus and Scaphechinus mirabilis . Polystyrene beads injected into the unfertilized egg were at a standstill in the protoplasm. After fertilization, these beads exhibited movements toward the center of the sperm aster along the rays, and finally gathered around the astral center. They were distributed in blastomeres together with the mitotic centers during successive cleavages. When injected into eggs during mitosis, beads moved to the centers of the mitotic asters along astral rays. The injected beads did not move when the aster was disorganized by treatment with Colcemid, and moved when it formed after UV-irradiation. These results indicate that microtubules of astral rays are essential to the movement of polystyrene beads. The movement of small polystyrene beads (0.2–0.3 μm in diameter) resembled the saltatory movement of endogenous cytoplasmic granules, and the movement of large beads (ca. 1 μm in diameter) resembled the female pronuclear migration. All of these movements observed in fertilized eggs were demonstrated to be microtubule-dependent, perhaps sharing the same basic mechanisms.     

Locomotive mechanism of Physarum plasmodia based on spatiotemporal analysis of protoplasmic streaming

We investigate how an amoeba mechanically moves its own center of gravity using the model organism Physarum plasmodium. Time-dependent velocity fields of protoplasmic streaming over the whole plasmodia were measured with a particle image velocimetry program developed for this work. Combining these data with measurements of the simultaneous movements of the plasmodia revealed a simple physical mechanism of locomotion. The shuttle streaming of the protoplasm was not truly symmetric due to the peristalsis-like movements of the plasmodium. This asymmetry meant that the transport capacity of the stream was not equal in both directions, and a net forward displacement of the center of gravity resulted. The generality of this as a mechanism for amoeboid locomotion is discussed.     

MIGRATORY CELL LOCOMOTION VERSUS NERVE AXON ELONGATION : Differences Based on the Effects of Lanthanum Ion

The effects of lanthanum ions (La⁺⁺⁺) on the locomotion and adhesion of g lial cells and elongating nerve axons are reported. La⁺⁺⁺ increases adhesion of both glia and of nerve growth cones to a plastic substratum. La⁺⁺⁺ also markedly reduces glia locomotion, but it does not inhibit nerve elongation. Electron-opaque deposits are seen on the cell surface and within cytoplasmic vesicles of glia and nerves cultured in a La⁺⁺⁺-containing medium. Possible modes of action for La⁺⁺⁺ are discussed, particularly the possibilities that Ca⁺⁺ fluxes or Ca⁺⁺ involvement in adhesion are altered by La⁺⁺⁺. The results are consistent with the hypothesis that cell migration and nerve axon elongation differ in mechanism, with respect to both adhesive interactions and the activity of microfilament systems.     

Elegant‐crested Tinamous Eudromia elegans do not synchronize head and leg movements during head‐bobbing

Head‐bobbing is the fore–aft movement of the head relative to the body during terrestrial locomotion in birds. It is considered to be a behaviour that helps to stabilize images on the retina during locomotion, yet some studies have suggested biomechanical links between the movements of the head and legs. This study analysed terrestrial locomotion and head‐bobbing in the Elegant‐crested Tinamou Eudromia elegans at a range of speeds by synchronously recording high‐speed video and ground reaction forces in a laboratory setting. The results indicate that the timing of head and leg movements are dissociated from one another. Nonetheless, head and neck movements do affect stance duration, ground reaction forces and body pitch and, as a result, the movement of the centre of mass in head‐bobbing birds. This study does not support the hypothesis that head‐bobbing is itself constrained by terrestrial locomotion. Instead, it suggests that visual cues are the primary trigger for head‐bobbing in birds, and locomotion is, in turn, constrained by a need for image stabilization and depth perception.     

Gross anatomy of the hindlimb skeletal system of theGalago senegalensis

The saltatory type of movement of the Prosimian,Galago senegalensis or Lesser Bushbaby is consistent with the gross anatomy of the skeletal system of the hindlimbs. The ilium and foot are elongated as in the tarsius, another prosimian having a similar mode of locomotion. The length of the foot is due largely to an elongated navicular and calcaneus. Other details of the complete bony structure of the hindlimbs are described and compared with animals of the suborders Prosimii and Anthropoidea when such is appropriate.Support for this study was supplied by the Easter Seal Research Foundation of the National Society for Crippled Children and Adults, Inc. and USPHS grant NS08590.     

Pure thoughts with impure proteins: Permeabilized cell models of organelle motility

Permeabilized cell models provide an experimental middle ground wherein the in vitro properties of mechanochemical proteins can be reconciled with the physical and topological constraints of the intact cell. Several well-studied examples of organelle motility are described here, including the actin-based cytoplasmic streaming of Characean algae, the microtubule-based aggregation and dispersion of pigment granules in chromatophores and the saltatory movements of vesicles along microtubules in fibroblasts and macrophages. The permeabilized models developed for these systems have helped to integrate observations in vivo with in vitro assays of motor proteins.