bluuebunny/crossref_metadata_2025_split huggingface dataset:
Direct observation of molecular motility by light microscopy
Authors: Harada Yoshie , Yanagida Toshio 
Published: February 2005

AbstractWe used video‐fluorescence microscopy to directly observe the sliding movement of single fluorescently labeled actin filaments along myosin fixed on a glass surface. Single actin filaments labeled with phalloidin‐tetramethyl‐rhodamine, which stabilizes the filament structure of actin, could be seen very clearly and continuously for at least 60 min in O2‐free solution, and the sensitivity was high enough to see very short actin filaments less than 40 nm long that contained less than eight dye molecules. The actin filaments were observed to move along double‐headed and, similarly, single‐headed myosin filaments on which the density of the heads varied widely in the presence of ATP, showing that the cooperative interaction between the two heads of the myosin molecule is not essential to produce the sliding movement. The velocity of actin filament independent of filament length (>1 μm) was almost unchanged until the density of myosin heads along the thick filament was decreased from six heads/14.3 nm to 1 head/34 nm. This result suggests that five to ten heads are sufficient to support the maximum sliding velocity of actin filaments (5 μm/s) under unloaded conditions. In order for five to ten myosin heads to achieve the observed maximum velocity, the sliding distance of actin filaments during one ATP cycle must be more than 60 nm.
Optical approaches to the study of foraminiferan motility
Authors: Travis Jeffrey L. , Bowser Samuel S. 
Published: February 2005

AbstractMicrotubules are the major cytoskeletal component of foraminiferan reticulopodia. Video‐enhanced differential interference contrast light microscopy has demonstrated that the microtubules serve as the intracellular tracks along which rapid bidirectional organelle transport and cell surface motility occurs. Microtubules appear to move, both axially and laterally within the pseudopodial cytoplasm, and these microtubule translocations appear to drive the various reticulopodial movements. F‐actin is localized to discrete filament plaques form at sites of pseudopod‐substrate adhesion. Correlative immunofluorescence and electron microscopy reveals a structural interaction between microtubules and the actin‐containing filament plaques. Our recent data on reticulopodial motility are discussed in an historical context, and a model for foram motility, based on motile microtubules, is presented.
Regulation of the distribution of carotenoid droplets in goldfish xanthophores and possible implication to secretory processes
Authors: Tchen T. T. , Lo Szecheng J. , Lynch Thomas J. , Palazzo Robert E. , Peng Gang , Walker Gary R. , Wu Bei‐Yui , Yu Fu‐Xin , Taylor John D. 
Published: February 2005

AbstractIn goldfish xanthophores, the formation of pigment aggregate requires: (1) that a pigment organelle (carotenoid droplet) protein p57 be in the unphosphorylated state; (2) that self‐association of pigment organelles occur in a microtubule‐independent manner; and (3) that pigment organelles via p57 associate with microtubules. In the fully aggregated state, the pigment organelles are completely stationary. Pigment dispersion is initiated by activation of a cAMP‐dependent protein kinase, which phosphorylates p57 and allows pigment dispersion via an active process dependent on F‐actin and a cytosolic factor. This factor is not an ATPase, and its function is unknown. However, its abundance in different tissues parallels secretory activity of the tissues, suggesting a similarity between secretion and pigment dispersion in xanthophores. The identity of the motor for pigment dispersion is unclear. Experimental results show that pigment organelles isolated from cells with dispersed pigment have associated actin and ATPase activity comparable to myosin ATPase. This ATPase is probably an organelle protein of relative molecular mass ∼72,000, and unlikely to be an ion pump. Isolated pigment organelles without associated actin have 5× lower ATPase activity. Whether this organelle ATPase is the motor for pigment dispersion is under investigation. The process of pigment aggregation is poorly understood, with conflicting results for and against the involvement of intermediate filaments.
Dynamics of the endoplasmic reticulum in living onion epidermal cells in relation to microtubules, microfilaments, and intracellular particle movement
Authors: Allen Nina Strömgren , Brown Douglas T. 
Published: February 2005

AbstractThe endoplasmic reticulum (ER) and associated organelle and particle movements in onion (Allium cepa) bulb scale epidermal cells were observed, recorded, and analyzed using computer‐assisted video (AVEC‐DIC, AVEC‐POL and fluorescence) microscopy. The ER is composed of two interconnected sets of filamentous membrane tubules with diameters ranging from 0.1 to 0.5 μm. The first form a more stable, stationary network of intersecting polygonal membrane tubules lying closely appressed to the plasma membrane and continuous with a second very dynamic set of longer membrane tubules that often are located parallel to each other, shifting rapidly around the cytoplasm and forming dynamic knots or organization centers. The ER, mitochondria, and spherosomes fluoresced upon chlortetracycline treatment and are therefore presumed to sequester calcium. ER and mitochrondria also stain with the fluorescent dye, rhodamine 123. Mitochrondria and spherosomes are seen to move in the cytoplasm only along paths parallel to the axis of the ER tubules. Smaller particles (0.5 μm) tend to follow these same paths but may occasionally move independently. Particles and organelles move in close, but not in direct, association with the ER tubules. In optically favored cells, actin filaments were occasionally recorded located in parallel with the ER tubules and directly associated with moving particles. Streaming ceased promptly and reversibly upon treatment with cytochalasin B, which did not visibly disrupt the ER. Short‐term treatment with colchicine did not inhibit streaming or disrupt the ER network, whereas long‐term (hours) colchicine treatments caused the disappearance of the stationary, cortical polygonal networks and an aggregation of still slowly moving organelles and particles onto now visible actin filaments. This suggests that microtubule breakdown disrupts the three‐dimensional distribution of the ER and rearranges actin filaments in the cell's cytoplasm. Actin filaments must be directly involved in generation of movement of the particles and organelles. A three‐dimensional model, based on optical sectioning of the epidermal cells, is proposed to illustrate the distribution of the endoplasmic reticulum in onion epidermal cell cytoplasm.
Microtubule dynamics in the chromosomal spindle fiber: Analysis by fluorescence and high‐resolution polarization microscopy
Authors: Cassimeris L. , Inoué S. , Salmon E. D. 
Published: February 2005

AbstractWe describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23–24°C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half‐time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half‐life of microtubule attachment to the kinetochore is less than several min at 23–24°C.In the second study, we used high‐resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150‐nm‐thick optical sections that microtubules throughout the spindle laterally associate for several sec into “rods” composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.
Three‐dimensional localization and redistribution of F‐actin in higher plant mitosis and cell plate formation
Authors: Molè‐Bajer Jadwiga , Bajer Andrew S. , Inoué Shinya 
Published: February 2005

AbstractThe distribution of F‐actin cables in dividing endosperm cells of a higher plant, Haemanthus, was visualized with the immunogold‐silver‐enhanced method and compared with the arrangement of immunogold‐stained microtubules in the same cells. The three‐dimensional distribution of F‐actin cables and microtubules during mitosis and cell plate formation was analyzed using ultrathin optical sectioning of whole mounts in polarized light video microscopy. F‐actin cables form a loose irregular network in the interphase cytoplasm. Much of this network remains outside of the spindle during mitosis. A few F‐actin cables were detected within the spindle. Their pronounced rearrangement during mitosis appears to be related to the presence and growth of microtubule arrays. During prometaphase, actin cables located on the spindle surface and those present within the spindle tend to arrange parallel to the long axis of the spindle. Cables outside the spindle do not reorient, except those at the polar region, where they appear to be compressed by the elongating spindle. Beginning with mid‐anaphase, shorter actin cables oriented in various directions accumulate at the equator. Some of them are incorporated into the phragmoplast and cell plate and are gradually fragmented as the cell plate is formed and ages. Actin cables adjacent to microtubule arrays often show a regular punctate staining pattern. Such a pattern is seldom observed in the peripheral cytoplasm, which contains few microtubules. The rearrangement of F‐actin cables mimicks the behavior of spindle inclusions, such as starch grains, mitochondria, etc., implying that F‐actin is redistributed passively by microtubule growth or microtubule‐related transport. Thus F‐actin or actomyosin‐based motility does not appear to be directly involved in mitosis and cytokinesis in higher plants.
Dynamics of microtubules visualized by darkfield microscopy: Treadmilling and dynamic instability
Authors: Hotani Hirokazu , Horio Tetsuya 
Published: February 2005

AbstractIndividual microtubules undergoing treadmilling in vitro were visualized by darkfield light microscopy, and the relationship between treadmilling and dynamic instability was studied as a function of microtubule‐associated proteins (MAPs). In order to demonstrate treadmilling directly by real‐time observation, we constructed three‐block microtubules, the center‐block of which was decorated with Tetrahymena dynein. The decorated block can easily be distinguished from undecorated blocks in the darkfield microscope because the decorated one appears much thicker. At steady‐state conditions, the length of an undecorated block at one end increased and that at another end decreased, while the decorated center‐block did not change in its length. The results from these direct observations show that calf brain 3X‐microtubules exhibit a treadmilling flux of 0.9 μm/h.Using a similar microscopy technique, we previously demonstrated that phosphocellulose PC‐microtubules existed in either the growing or the shortening phase and alternated quite frequently at steady‐state conditions (dynamic instability). How does treadmilling relate to dynamic instability? An image recording of individual 3X‐microtubules containing MAPs revealed that the microtubules undergo treadmilling and do not exhibit any dynamic instability. This evidence shows that MAPs suppress the dynamic instability of microtubules. That is, treadmilling can take place in the steady state only after microtubules have been stabilized by MAPs.
Detection of single fluorescent microtubules and methods for determining their dynamics in living cells
Authors: Sammak Paul J. , Borisy Gary G. 
Published: February 2005

AbstractThe ability to tag biological molecules fluorescently and to detect their distribution in living cells has promoted the study of cytoplasmic organization in general and microtubule dynamics in particular. The techniques that we have selected and developed allowed the determination of spatial and temporal changes of the microtubule network in living fibroblasts at the level of individual microtubules. We have employed two general approaches for determining pattern changes: direct video microscopy and photobleaching and subsequent observation. Direct observation of fluorescent microtubules by high‐definition video microscopy provided good spatial resolution at several time points, but was limited to the less congested and thinner periphery of the cell. This approach was made possible by a relatively bright, photostable reporter, xrhodamine‐tubulin, and showed that microtubules underwent rounds of assembly and disassembly from their ends. Bleaching and subsequent observation of lysed cells improved the signal to noise ratio by extracting soluble chromophore and permitted observations in congested areas, but was limited to a single time interval. This approach demonstrated that microtubule domains were replaced one by one and that turnover was most rapid at the cell periphery. Antibodies specific for nonbleached chromophore can be used to enhance the signal to noise ratio further or to extend spatial resolution by the use of immunoelectron microscopy. Direct video microscopy and photo‐bleaching are two approaches to the study of dynamics that have complementary strengths and wide application to the biology of living cells.
Vesikin, a vesicle associated ATPase from squid axoplasm and optic lobe, has characteristics in common with vertebrate brain MAP 1 and MAP 2
Authors: Do Cuong V. , Sears Edward B. , Gilbert Susan P. , Sloboda Roger D. 
Published: February 2005

AbstractVesikin, a protein that can associate with squid axoplasmic vesicles or optic lobe microtubuies, has been implicated as a force‐generating molecule involved in microtubule‐dependent vesicle transport [Gilbert and Sloboda, 1986, 1988]. Because vesikin crossreacts with an antibody to porcine brain microtubule associated protein 2 (MAP 2), studies were conducted to compare squid vesikin and brain MAPs. When taxol stabilized microtubules containing vesikin as a microtubule associated protein were incubated in the presence of ATP, vesikin dissociated from the microtubule subunit lattice. This behavior would be expected for an ATP‐dependent, force generating molecule that serves as a crossbridge between vesicles and microtubules. When chick brain microtubules were treated under the same conditions, MAP 2 remained bound to the microtubules while MAP 1 dissociated in a manner similar to vesikin. One dimensional peptide mapping procedures revealed that, although digestion of vesikin and MAP 2 generated several peptides common to both proteins, vesikin and MAP 2 are clearly not identical. Furthermore, the addition of vesikin or MAPS 1 and 2 to purified tubulin stimulated microtubule assembly in a manner dependent on the concentration of added protein. These findings demonstrate that brain MAPs share characteristics common to squid vesikin and support the suggestion that brain MAPs 1 and 2 might act as a force generating complex for vesicle transport in higher organisms.
Dynamic instability and motile events of native microtubules from squid axoplasm
Authors: Weiss Dieter G. , Langford George M. , Seitz‐Tutter Dieter , Keller Franz 
Published: February 2005

AbstractNative microtubules from extruded axoplasm of squid giant axons were used as a paradigm to characterize the motion of organelles along free microtubules and to study the dynamics of microtubule length changes. The motion of large round organelles was visualized by AVEC‐DIC microscopy and analyzed at a temporal resolution of 10 frames per second. The movements were smooth and showed no major changes in velocity or direction. During translocation, the organelles paused very rarely. Superimposed on the rather constant mean velocity was a velocity fluctuation, which indicated that the organelles are subject to considerable thermal motion during translocation. Evidence for a regular low‐frequency oscillation was not found. The thermal motion was anisotropic such that axial motion was less restricted than lateral motion. We conclude that the crossbridge connecting the moving organelle to the microtubule has a flexible region that behaves like a hinge, which permits preferential movement in the direction parallel to the microtubule. The dynamic changes in length of native microtubules were studied at a temporal resolution of 1 Hz. About 98% of the native microtubules maintained their length (“stable” microtubules), while 2% showed phases of growing and/or shrinking typical for dynamic instability (“dynamic” microtubules). Gliding and organelle motion were not influenced by dynamic length changes. Transitions between growing and shrinking phases were low‐frequency events (1–10 minutes per cycle). However, a new type of microtubule length fluctuation, which occurred at a high frequency (a few seconds per cycle), was detected. The length changes were in the 1–3 μm range. The latter events were very prominent at the (+) ends. It appears that the native axonal microtubules are much more stable than the purified microtubules and the microtubules of cultured cells that have been studied thus far. Potential mechanisms accounting for the three states of microtubule stability are discussed. These studies show that the native microtubules from squid giant axons are a very useful paradigm for studying microtubule‐related motility events and microtubule dynamics.
Microtubule gelation‐contraction in vitro and its relationship to component a of slow axonal transport
Authors: Weisenberg Richard C. , Flynn James , Gao Baochong , Awodi Shuaibo 
Published: February 2005

AbstractMicrotubule proteins, isolated by cycles of assembly, will undergo ATP‐dependent gelation‐contraction in vitro. A particulate component is present in these preparations, which is required for the gelation‐contraction of microtubules assembled from purified tubulin. These particulates contain tubulin, neurofilament, spectrin, MAP2, and other as yet unidentified proteins. The particulates have a microtubule‐stimulated ATPase that may be unique and is the likely motor for microtubule gelation‐contraction. The basic structural unit of these particulates appears to be a crescent‐shaped, or hemispherical, granule about 20 nm in diameter. The particles move along microtubule walls at a rate of about 1 μm. When compared to known physiological phenomena, microtubule gelation‐contraction has striking similarities to component a of slow axonal transport (SCa), but displays no relationship to slow component b or to fast transport. On the basis of their similarities in composition, solubility, and rate of movement, we have proposed that the particulates responsible for microtubule gelation‐contraction are the insoluble protein complexes, which have been suggested to be the transported component of SCa. We have termed these structures “slow component a particulates” or “SCAPs.” It is probable that similar motile protein complexes exist in cells other than neurons, and we propose the term “dynasome” to describe such structures in general.
Modulation of nuclear rotation in neuronal interphase nuclei by nerve growth factor, by γ‐aminobutyric acid, and by changes in intracellular calcium
Authors: Fung Leslie C. , De Boni Umberto 
Published: February 2005

AbstractNuclear rotation (NR) is typically measured as motion of nucleoli within nuclei of cells in vitro. This occurs in cycling cells. However, its observation in neurons arrested in interphase indicates that mechanisms related to mitosis are not a prerequisite. We have recently shown that NR occurs in three dimensions within the nuclear space, that it occurs within the space delineated by the outer nuclear membrane and that it includes chromatin domains in addition to nucleoli and have postulated that this motion of chromatin domains is related to changes in gene expression. We now show that exposure of dorsal root, sensory neurons in vitro to nerve growth factor (NGF) or to γ‐aminobutyric acid (GABA), agents which alter gene expression, and to agents causing redistribution of calcium, such as EGTA and the calcium ionophore A23187, significantly alters NR. The NGF increased the mean rate of NR and did so at a time after exposure when activity of RNA polymerases have been shown to rise. Exposure to GABA resulted, within minutes, in shifts of the nucleolus within the three‐dimensional space of the nucleus, associated in some neurons with significant, sigmoidal increases in the rate of NR. The calcium ionophore A23187 as well as chelation of extracellular calcium with EGTA similarly increased rates. Importantly, excess calcium, with EGTA remaining present, returned NR of all nucleoli to rates not different from controls. This indicates that the increase in NR seen with EGTA is specific to the chelation of calcium and not a nonspecific response to EGTA. It is difficult to link the action of agents which alter gene expression or transmembrane ion balance with changes in NR. Nevertheless, in support of our hypothesis, the results presented here show that agents known to alter gene expression, alter NR in a temporally coincident manner and that they do so, possibly, by calcium‐dependent mechanisms.
Calcium regulation of flagellar curvature and swimming pattern in triton X‐100–extracted rat sperm
Authors: Lindemann Charles B. , Goltz Jason S. 
Published: February 2005

AbstractFree Ca2+ changes the curvature of epididymal rat sperm flagella in demembranated sperm models. The radius of curvature of the flagellar midpiece region was measured and found to be a continuous function of the free Ca2+ concentration. Below 10−7 M free Ca2+, the sperm flagella assumed a pronounced curvature in the same direction as the sperm head. The curvature reversed direction at 2.5 × 10−6 M Ca2+ to assume a tight, hook‐like bend at concentrations of 10−5 to 10−4 M free Ca2+. Sodium vanadate at 2 × 10−6 M blocked flagellar motility, but did not inhibit the Ca2+‐mediated change in curvature. Nickel ion at 0.2 mM and cadmium ion at 1 μM interfered with the transition and induced the low Ca2+ configuration of the flagellum. The forces that maintain the Ca2+‐dependent curvature are locally produced, as dissection of the flagella into segments did not significantly alter the curvature of the excised portions. Irrespective of the induced pattern of curvature, the sperm exhibited coordinated, repetitive flagellar beating in the presence of ATP and cAMP. At 0.3 mM ATP the flagellar waves propagated along the principal piece while the level of free Ca2+ controlled the overall curvature. When Ca2+‐treated sperm models with hooked midpieces were subjected to higher concentrations of ATP (1–5 mM), some cells exhibited a pattern of movement similar to hyperactivated motility in capacitated live sperm. This type of motility involved repetitive reversals of the Ca2+‐induced bend in the midpiece, as well as waves propagated along the principal piece. The free Ca2+ available to the flagellum therefore appeared to modify both the pattern of motility and the flagellar curvature.