bluuebunny/crossref_metadata_2025 huggingface dataset:
Motility, cell shape, and locomotion of neutrophil granulocytes
Authors: Keller H. U.
Published: February 2005

Activation of the motile apparatus by chemokinetic factors cannot be reliably assessed in cells that are attached to a solid substratum because motility can be totally abolished by excessive adhesion. It is however, necesary to quantify the activation of the motile apparatus in order to analyze and understand chemokinetic responses.

It was the purpose of the present work to establish morphological criteria that can be used to quantify motility in nonadherent (floating) neutrophils and to predict the locomotor response under conditions of limited adhesion. The proportion of neutrophils performing crawling‐like movements (polarized cells) in suspension correlates very closely with stimulated locomotion at low to optimal concentration of f‐Met‐Leu‐Phe, ie, under conditions of limited adhesion. Reduced locomotion at supraoptimal concentrations of f‐Met‐Leu‐Phe has also morphological correlates. The major feature is the decrease in the proportion of neutrophils performing crawling‐like movements and the corresponding appearance of cells that are motile but not polarized in suspension and that do not locomote on the substratum. Concentration‐dependent changes in neutrophil length and in the proportion of polarized neutrophils with and without tail were also observed. The locomotor potential of neutrophils under conditions of limited contact with the substratum can be predicted on the basis of their motile behavior, in particular the proportion of cells showing crawling‐like movements, in suspension. In combination with measurements of adhesion the procedure should permit a more complete analysis of the regulation of chemokinetic responses.
Coelomocyte motility
Authors: Edds K. T. , Chambers C. , Allen R. D.
Published: February 2005

We have utilized a video‐enhanced contrast system coupled to a DIC‐equipped microscope to examine the motility of both whole coelomocytes and individual filopodia. When the cells are left in diluted coelomic fluid, they exhibit a fibroblast‐like mode of translocation across the substrate. These cells extend lamellipodia at their advancing margin and develop retraction fibers at the trailing edge. Filopodia are actively extended from the lamellipodia of the advancing margin. Cells that are washed free of the coelomic fluid and placed in an isotonic buffer lose their ability to translocate. Filopodia on these stationary cells are seen to undergo a series of waving and bending motions. These motions are rapid and result in a filopodium folding back upon itself only to reextend later. Both forms of motility are discussed in light of the existing structural and biochemical knowledge of this and other cell types.
Ciliary but not saltatory movements are inhibited by vanadate microinjected into living cultured cells
Authors: Buckley Ian , Stewart Murray
Published: February 2005

To test the idea that saltatory organelle movements of nonmuscle cells might be driven by microtubule‐dynein interactions, we microinjected vanadate into several different types of cultured cell. Solutions of sodium metavanadate made up in a simple buffered salt solution were pressure microinjected into fully spread cells in an open‐topped culture chamber placed on the stage of an inverted microscope. The cells were observed by oil‐immersion phase‐contrast optics and results were recorded on movie film. Vanadate, at 10−5‐10−2 M, microinjected into cultured chick embryo fibroblasts, failed to inhibit organelle movements. To test the effectiveness of vanadate's inhibitory action under living cell conditions, ciliated epithelial cells were micro‐injected. In these cells even the smallest microinjection of 5 × 10−5 M vanadate caused an immediate cessation of ciliary beating. Moreover, in cells that were well spread it was found that whereas vanadate, at 5 × 10−5 × 10−3M, inhibited ciliary motion, it failed to inhibit organelle saltations in the same cell. To determine whether vanadate would inhibit a living actin‐myosin system, myocardial cells were also microinjected. Following microinjection of 5 × 10−5 and 5 × 10−4M vanadate a temporary tonic contraction (which also occurred following microinjection of buffer alone) was followed by regular beating. Taken together these results demonstrate that in living cell systems microtubule‐dynein interactions are as sensitive to vanadate inhibition as they are in demembranated model systems, and that a working actin‐myosin system in a living muscle cell does not share this great sensitivity. In light of the pronounced differential inhibitory effects of vanadate on the movements of cilia and organelles, our results suggest that saltatory organelle movements in chick embryo fibroblasts and rabbit oviduct epithelial cells are unlikely to be brought about by microtubule‐dynein interactions.
The interaction of cAMP with modeled bull sperm
Authors: Lindemann Charles B. , Lipton Melissa , Shlafer Roman
Published: February 2005

Demembranated and membrane disrupted bull sperm models exhibit an increase in motility when exposed to cAMP. Tritium‐labeled cAMP was used to locate the initial site of action of cAMP in the modeled sperm preparations. cAMP did not bind selectively to the modeled cells, and the presence or absence of plasma membrane fragments on the models did not significantly alter this result. When suspension medium taken from modeled sperm preparations was subjected to gel filtration on Sephadex G25‐150 columns, cAMP bound to a high molecular weight component that eluted with the void volume. The responsible binding factor is a soluble component that is released when the plasma membranes of the sperm are disrupted during the modeling procedure. To test the importance of the cAMP binding factor, modeled sperm were centrifuged, the super‐natant solution was decanted, and the cells were resuspended in fresh medium. After this treat‐ment the cells could be restored to motility with Mg‐ATP but no longer exhibited a response to cAMP. Furthermore, addition of cAMP binding factor isolated by gel filtration partially restored the response of these sperm to cAMP. Investigation of the properties of the cAMP‐binding factor have confirmed that it is specific for cAMP, with a much lower affinity for AMP and cGMP. In the pre‐sence of a large excess of unlabeled cAMP the labeled complex has a half‐life of approximately 1 hour. Our results indicate that the action of cAMP on the motility of modeled sperm is mediated by its attachment to a high molecular weight, soluble component of the cell cytoplasm.
Cell movement and its coordination in swarms of myxococcus xanthus
Authors: Kaiser Dale , Crosby Cathy
Published: February 2005

The coordinated movement of many cells—a process called swarming—permits myxobacteria to spread rapidly over a surface. We have investigated the mechanism of swarming in Myxococcus xanthus by making time‐lapse motion pictures and by measuring the dependence of cell movement and spreading rate on the concentration of cells. Motion pictures of spreading zones showed that spreading resulted from motility, not growth, and that a swarm spread outward by establishing a loose reticulum of cells, then later filling it in. The spreading rate of wildtype strains was found to be highly dependent on cell density, increasing about 8‐fold as the cell density was increased from 2.5 to 200 units. Mutants swarmed if they possessed only the A‐motile component (A+S−) or only the S‐motile component (A−S+) of wild type (A+S+); their spreading rate increased with cell density but was always less than A+S+. Individual A+S+, A+S−, and A−S+ cells executed typical gliding movements and (when moving) progressed at approximately the same speed, as if A and S motility were different ways of engaging the same gliding machine. Photographic studies of an A−S+ strain showed that cells moved only if they were separated by less than approximately one cell length from each other. This provided further evidence that pili, which are present on A+S+ and A−S+ cells and which extend about one cell length, could be responsible for switching on movement in S‐motile cells, and presumably in wild type as well.
The effects of taxol on cytoskeletal components in cultured fibroblasts and epithelial cells
Authors: Green Kathleen J. , Goldman Robert D.
Published: February 2005

Taxol promotes microtubule (MT) assembly in vitro and induces the reorganization of the cytoskeleton into unusual MT arrays in cultured cells. The possibility that taxol also has an indirect effect on intermediate filaments (IF) was investigated. In baby hamster kidney (BHK‐21) and human skin (ENSON) fibroblasts treated with 1–10 μM taxol for 1–24 h, the drug induces changes which are similar to those produced by colchicine. These include a loss of major cellular extensions, a redistribution of organelles to a perinuclear location, and an inhibition of locomotion. Saltatory particle movements are not inhibited, however. Ruffling and filopod formation continue, indicating that cells are viable up to 24 h.

Polarized light microscopy of living fibroblasts treated with taxol reveals the presence of perinuclear birefringent material which has been examined by immunofluorescence. In control cells, IF and MT radiate from a juxtanuclear region and extend to the cell periphery. In taxol‐treated cells, MT and IF are excluded from cell margins, forming large central bundles.

In the epithelial cell lines PtK2 and PAM, the keratin system of IF does not become redistributed; in PtK2, however, a second fibroblastlike system of IF does become redistributed to a perinuclear position during taxol treatment.

Ultrastructural analyses show that taxol‐treated fibroblasts contain parallel arrays of cross‐bridged MT‐IF as well as bundles of MT exclusive of IF. Epithelial cells contain a predominance of IF‐free MT bundles which are organized into hexagonally packed arrays. In these bundles MT frequently exhibit hooks or other incomplete MT profiles and are linked by filamentous material.
Differential response to contact during embryonic nerve‐nonnerve cell interactions
Authors: Nuttall Robert P. , Zinsmeister Philip P.
Published: February 2005

The outcome of contact interactions involving neurons and nonneurons varies depending on the cell types involved. When neuronal growth cones from either ciliary (motor) or dorsal root (sensory) ganglia directly contact the lamellipodium of an embryonic heart fibroblast, both neurite elongation and fibroblast locomotion are inhibited. This occurs in spite of the fact that cell‐surface activity in both cells continues unabated. Such contact inhibition is not observed when homologous ganglionic nonneurons are involved in the interaction. In fact, these cells become intimately associated with growth cones and/or neuritic shafts as a result of the contact. The detailed nature of the respose to contact exhibited by nerves and nonnerves varies not only with cell type but also with the portion of the cell involved in the contact. Growth cone filopodia tend to actively palpate the fibroblast surface, whereas spread regions, termed “veils,” form areas of apposition with fibroblast lamellipodia. This latter situation resembles the “typical” contact inhibition of locomotion that occurs following embryonic heart fibroblast‐fibroblast interactions. Growth cones also frequently exhibit contact guidance when interacting with nonruffling lateral surfaces of heart fibroblasts.
A quantitative comparison of cellular motile systems
Authors: Nicklas R. Bruce
Published: February 2005

Cellular motile systems as diverse as muscle and the mitotic spindle have been compared by their specific power output: the maximum power they develop per unit of engine volume. Striated muscles and flagella have high specific output; their performance is comparable to that of typical automobile engines. The cytokinetic furrow and the mitotic spindle have very much lower specific power output. The furrow's output is 7,000 times lower than muscle and the spindle's is 300,000 times lower. Different macromolecules have been used to generate power in systems with similar output (muscles and flagella) and, conversely, the same macromolecular motor has been used in systems with very different output (muscles and cytokinetic furrows). The common feature amid this diversity is adaptation to a particular biological role, which specific power output reflects very well. High values are found where a powerful, compact engine should be advantageous, while low values are found where precision, not power, matters most.
A flagellar surface glycoprotein mediating cell‐substrate interaction in Chlamydomonas
Authors: Bloodgood Robert A. , Workman Lisa J.
Published: February 2005

The Chlamydomonas flagellar surface exhibits interesting adhesive properties that are associated with flagellar surface motility. This dynamic surface property can be exhibited as the binding and movement of small polystyrene microspheres or as the interaction of the flagellar surface with a solid substrate followed by whole cell locomotion, termed “gliding.” In order to identify flagellar surface proteins that mediate substrate interaction during flagellar surface motility, two immobilized iodination systems were employed that mimic the conditions for flagellar surface motility: small polystyrene microspheres derivatized with lactoperoxidase, and large glass beads derivatized with Iodogen. Use of these iodination conditions resulted in preferential iodination of a high‐molecular‐weight glycoprotein with apparent molecular weight of 300,000–350,000. These results suggest this glycoprotein as a major candidate for the surface‐exposed adhesive component that directly interacts with the substrate and couples the substrate to a system of force transduction presumed to be located within the flagellum.
Selective immunocytochemical detection of fluorescent analogs with antibodies specific for the fluorophore
Authors: Luby‐Phelps Katherine , Amato Philip A. , Taylor D. Lansing
Published: February 2005

Fluorescent analogs of cellular components are finding increasing use in the field of cell biology. The power of this technique can be augmented by the use of antibodies specific for the fluorophore to visualize selectively the fluorescent analog at the electron microscope level. Rabbit antibodies specific for fluorescein were elicited and purified according to published methods (Lopatin and Voss [1971]: Biochemistry 10:208). Immune sera and IgG formed precipitin lines with fluorescein‐labeled proteins in Ouchterlony immunodiffusion assays, and significantly quenched the fluorescence of fluorescein‐labeled proteins. Immune IgG and Fab fragments decorated fluorescein‐labeled actin, but not unlabeled actin, in negative‐stained preparations. Anti‐fluorescein IgG was used for immunofluorescent localization of fluorescein‐labeled actin following microinjection of the fluorescent analog into living cells. This approach was extended to the immunoelectron microscopic localization of the injected analog at the subcellular level by the use of an electron‐dense marker coupled to goat anti‐rabbit IgG. Many other fluorescent probes also can be used as haptens for production of antibodies. Therefore, a general method for localizing fluorescently labeled molecules at the electron microscopic level is now available. Several other applications of anti‐fluorescein antibody in studies involving fluorescent analogs are also suggested.
Three‐dimensional bend propagation in hamster sperm models and the direction of roll in free‐swimming cells
Authors: Yeung Ching‐Hei , Woolley David M.
Published: February 2005

Iontophoretic application of ATP to the flagellum of the demembranated hamster spermatozoon produced a planar pair of bends at the two ends of the stimulated site. During bend propagation, torsion appeared in the vicinity of the interbend in some responses such that the distal bend was twisted clockwise when viewed from the base of the flagellum. This pattern of propagation is consistent with the instantaneous configurations of free‐swimming cells previously described. The technique used here establishes that the three dimensionality arises from propagation per se, and does not depend on forces developed during swimming. The rolling of both free‐swimming intact and demembranated spermatozoa was examined by two‐color darkground videomicroscopy and the direction of rotation was, as predicted, always anticlockwise. A hypothetical mechanism, involving differential speeds of propagation of active sliding within the active microtubule subset, is proposed to account for the observed waveforms.
Mutants in paramecium tetraurelia defective in their axonemal response to calcium
Authors: Hinrichsen Robert D. , Saimi Yoshiro , Hennessey Todd , Kung Ching
Published: February 2005

Six mutants of Paramecium tetraurelia, which display altered axonemal responses to Ca++, are described. The mutants, designated atalantas, are impaired in their ability to swim backward when stimulated by ions or heat; instead they spin very rapidly in one place. Three mutants, ataA1–3, are completely unable to swim backward. The three lines, however, can be distinguished from one another by their forward swimming velocities. The remaining three mutants are leaky. ataB swims backward briefly when stimulated, then stops and spins in place. ataC and ataD are extremely leaky and only display the spinning phenotype at elevated temperatures. An electrophysiological analysis reveals that all six mutants have normal membrane properties, including the Ca++ inward current under voltage clamp. When the membrane is disrupted so as to allow the axoneme free access to Ca++, wild‐type cells swim backward, but the mutants do not. These data indicate the site(s) of lesion in the mutants is in the axoneme or in some step linking Ca++ influx and the axoneme, not within the ciliary membrane. These mutants may be useful in investigating the role of Ca++ in the regulation of axonemal motion.
Ca2+‐dependent contraction of human lung fibroblasts treated with triton X‐100: A role of Ca2+‐calmodulin‐dependent phosphorylation of myosin 20,000‐dalton light chain
Authors: Masuda Hirohisa , Owaribe Katsushi , Hayashi Hiroshi , Hatano Sadashi
Published: February 2005

Human lung fibroblast MRC‐5 cells treated with Triton X‐100 (MRC‐5 cell models) were able to contract in the presence of MgATP and Ca2+ of more than 1 μM. Immunofluorescence microscopy with antibodies to actin and myosin 20,000‐dalton (20 Kd) light chain revealed that stress fibers were prominent in MRC‐5 cell models. Use of a fluorescent actin probe, 7‐nitrobenz‐2‐oxa‐1,3‐diazole‐phallacidin permitted visualization of contraction of the stress fibers in the presence of MgATP and Ca2+. Of the proteins in MRC‐5 cell models, only a myosin 20 Kd light chain was phosphorylated in a Ca2+‐dependent manner. This Ca2+‐dependent phosphorylation of the 20 Kd light chain closely corresponded with the contraction of MRC‐5 cell models: 1) Both phosphorylation of the 20 Kd light chain and contraction of MRC‐5 cell models were inhibited by calmodulin antagonists such as N‐(6‐aminohexyl)5‐chloro‐1‐napthalene sulfonamide. 2) The threshold Ca2+ concentration for phosphorylation of the 20 Kd light chain was similar to that for contraction of MRC‐5 cell models. Both were lowered by exogenous calmodulin in a concentration‐dependent manner. 3) The 20 Kd light chain was thiophosphorylated by incubation of MRC‐5 cell models with an ATP analogue, adenosine 5′‐0‐(3‐thiotriphosphate) only in the presence of Ca2+. After this treatment, MRC‐5 cell models lost the Ca2+‐dependence for contraction. These results indicate that Ca2+‐calmodulin‐dependent phosphorylation of myosin 20 Kd light chain is required for contraction of MRC‐5 cell models.
Axonal elongation as a stochastic walk
Authors: Katz Michael J. , George Edwin B. , Gilbert Leslie J.
Published: February 2005

A new formula calculates rates of directed axonal growth (elongation or retraction) using measurements of growth cone movements. By explicitly separating changes in axonal length from other nonelongational growth cone movements, the calculated rates reflect the detailed cellular growth mechanisms more directly than previous growth measures. In addition, the formula produces three distinct parameters of axonal elongation: n, a growth step rate; s, a growth step size; and P, a probability that a growth step leads to axonal elongation. For normal and regenerating individual chick and frog axons in culture, the formula has quantitated the following differences: the axon itself can elongate more rapidly in the chick, and the axon elongates in smaller steps in the chick. The underlying dynamics of growth of regenerating axons are quite similar to normal axons, but, in the short term, regenerating axons elongate in larger steps and at a slower rate. The distribution of these new rate measurements suggests that the elongation of axons can be usefully modelled as a one‐dimensional stochastic walk.
Formation of myofibrils in spreading chick cardiac myocytes
Authors: Sanger Joseph W. , Mittal Balraj , Sanger Jean M.
Published: February 2005

Cardiac myocytes were isolated from 5–6‐day‐old chick embryos and allowed to spread in culture. The distribution of alpha‐actinin in the cells was followed for five days in culture by exposing permeabilized cells to rhodamine‐labeled alpha‐actinin and also by injecting the labeled alpha‐actinin into living myocytes. In addition to labeling the Z bands of sarcomeres, the added alpha‐actinin also labeled small particles that were usually arranged periodically in linear arrays with a spacing between particles of 0.3–2.0 μm. Actin was localized between the particles of alpha‐actinin by means of fluorescein‐labeled heavy meromyosin. The punctate localization of alpha‐actinin was prominent in pseudopods, behind ruffles, and at the periphery of spreading cells. Long rows of particles of alpha‐actinin were often parallel to one another with the alpha‐actinin particles in register. These linear arrays appeared to merge laterally to form strands with broader concentrations of alpha‐actinin. Other linear arrays were parallel to myofibrils in the cell and some extended outward from the ends of myofibrils. We conclude that during spreading of cardiac myocytes, myofibrils form at the cell periphery behind the extending margins of the cell, and that the aggregates of alpha‐actinin found in these areas are nascent Z bands in the forming myofibrils.
Analysis of the morphology and function of primary cilia in connective tissues:A cellular cybernetic probe?
Authors: Poole C. Anthony , Flint Michael H. , Beaumont Brent W.
Published: February 2005

More than 300 primary cilia have been identified electronmicroscopically in a variety of embryonic and mature connective tissue cells. To further define the enigmatic function of these cilia, we examined the interrelationships between the basal apparatus and cytoplasmic organelles and the ciliary shaft and the extracellular matrix. The basal diplosome was consistently associated with the secretory organelles including the maturing face of the Golgi complex, Golgi vacuoles and vesicles, the microtubular network, the plasma membrane, and coated pits and vesicles. Small vesicles and amorphous granules were also observed within the ciliary lumen and adjacent to the ciliary membrane. Microtubule‐membrane bridges linked axonemal tubules to the ciliary membrane. The position, projection, and orientation of the axoneme were influenced by the structural organisation and mechanical properties of the matrix and frequently caused angulation of the ciliary shaft relative to the basal body. Located midway between the secretory apparatus and the extracellular matrix, primary cilia would appear ideally situated to mediate the necessry interaction between the cell and its surrounding environment prerequisite to the formation and maintenance of a functionally effective matrix. We propose that primary cilia in connective tissue cells could act as multifunctional, cellular cybernetic probes, receiving, transducing, and conducting a variety of extrinsic stimuli to the intracellular organelles responsible for effecting the appropriate homeostatic feedback response to changes in the extracellular microenvironment.
Bending patterns of Chlamydomonas flagella: III. A radial spoke head deficient mutant and a central pair deficient mutant
Authors: Brokaw C. J. , Luck D. J. L.
Published: February 2005

Flash photomicrography at frequencies up to 300 Hz and computer‐assisted image analysis have been used to obtain parameters describing the flagellar bending patterns of mutants of Chlamydomonas reinhardtii. All strains contained the uni1 mutation, to facilitate photography. The radial spoke head deficient mutant pf17, and the central pair deficient mutant, pf15, in combination with suppressor mutations that restore motility without restoring the ultrastructural or biochemical deficiencies, both generate forward mode bending patterns with increased shear amplitude and decreased asymmetry relative to the “wild‐type” uni1 flagella described previously. In the reverse beating mode, the suppressed pf17 mutants generate reverse bending patterns with large shear amplitudes. Reverse beating of the suppressed pf15 mutants is rare. There is a reciprocal relationship between increased shear amplitude and decreased beat frequency, so that the velocity of sliding between flagellar microtubules is not increased by an increase in shear amplitude. The suppressor mutations alone cause decreased frequency and sliding velocity in both forward and reverse mode beating, with little change in shear amplitude or symmetry.
Proteins closely similar to flagellar tektins are detected in cilia but not in cytoplasmic microtubules
Authors: Amos W. B. , Amos L. A. , Linck R. W.
Published: February 2005

Affinity‐purified antibodies against Strongylocentrotus purpuratus sperm flagellar tektin polypeptides have been tested for cross‐reactivity with microtubules isolated from various sources, using indirect immunofluorescent staining and antibody binding to nitrocellulose replicas of SDS polyacrylamide gels. The antitektins reacted with sperm tail axonemes from four genera of sea urchins and with cilia from sea urchin embryos. Antibody binding was observed only if the specimens were prefixed by methods that would not preserve them well at an ultrastructural level. However, even after such fixation regimes, no antibody binding was detected to cytoplasmic microtubule arrays in the same embryos, to mitotic spindles isolated from sea urchin or to gill cilia from a mollusc. We conclude that, if tektins are present in sea urchin egg cytoplasmic microtubules, they are sufficiently different from the sperm tektins to have no common strongly antigenic determinants.
Tetrahymena cell model exhibiting Ca‐dependent behavior
Authors: Izumi Akemi , Miki‐Noumura T.
Published: February 2005

Using Tetrahymena pyriformis, strain w, and Tetrahymena thermophila, B‐1868, we prepared cell models that showed ciliary reversal with change in Ca‐ion concentration, as was also noted for the Paramecium cell model. No differences could be found between these two strains in the reactivation state, and their response to environmental conditions was essentially the same. The reactivation rate was 90% or more. Swimming velocity of the cell model was found to be 200 ± 49 μm/sec at 25.0°C ± 0.5°C, while the velocity for the living cells was 527 ± 101 μm/sec. Swimming velocity with change in environmental conditions, such as pH, Mg‐ATP, and Ca‐ion concentrations, was studied. Compared to the cell model of Paramecium, the Tetrahymena cell model had higher sensitivity toward Ca‐ion in the reactivation medium. The effects of chlorpromazine, and inhibitor of calmodulin, on the swimming behavior of the cell models were studied.
Chromosome movement during meiotic prophase in crane‐fly spermatocytes: III. Microtubules and the effects of Colcemid, nocodazole, and vinblastine
Authors: LaFountain James R.
Published: February 2005

The effects of Colcemid, nocodazole, and vinblastine on microtubules and on the movement of chromosomes during late diakinesis were investigated in spermatocytes of the crane fly Nephrotoma suturalis. The kinds of movements observed in untreated cells—sex bivalent rotations, sex bivalent excursions, and rotations and positional changes of autosomal bivalents—also were observed in drug‐treated cells. These results were obtained in living cells in which the disruption and inhibition of microtubule assembly had been confirmed with polarized light microscopy. Effects of Colcemid and nocodazole also were assessed in fixed cells using electron microscopy. The results are in agreement with a hypothesis that microtubules are not a force‐generating component of the molecular machinery that brings about prophase movements.
Use of multiple monoclonal antibodies to characterize the major microtubule‐associated protein in sea urchin eggs
Authors: Bloom George S. , Luca Francis C. , Collins Christine A. , Vallee Richard B.
Published: February 2005

Microtubules assembled from sea urchin eggs with the use of taxol contain a 77,000‐dalton protein as the major nontubulin component [Vallee and Bloom (1983): Proc Natl. Acad. Sci. U.S.A. 80:6259–6263]. We have raised five monoclonal antibodies to this protein to aid in its characterization. Immunoblot analysis of the sea urchin microtubule purification fractions indicated that the protein copurified quantitatively with microtubules. All five antibodies stained the mitotic spindle of dividing sea urchin eggs by immunofluorescence microscopy, indicating that the protein was a component of the mitotic spindle and suggesting that it was actually localized on microtubules in vivo. Immunofluorescent staining of higher resolution was observed in a subpopulation of the coelomic cells found in adult sea urchins, confirming that the 77,000‐dalton protein is indeed present on microtubules in vivo. Because taxol was not used for the immunofluorescence experiments, we conclude that the microtubule‐associated protein (MAP)‐like behavior of the 77,000‐dalton protein in vitro was not induced artifactually by taxol. To determine whether this protein is a component of sea urchin microtubules in general, cilia obtained from blastula stage embryos and sperm tail flagella were analyzed with the antibodies. The protein was undetectable by both immunoblot analysis and immunofluorescence microscopy in both preparations of axonemal microtubules. These results indicated that the 77,000‐dalton MAP is restricted to cytoplasmic and mitotic microtubules in the sea urchin. Furthermore, in view of its particular abundance in embryos, whose microtubules are devoted substantially to mitosis, the 77,000‐dalton MAP is likely to play an important role in regulating the activity of mitotic spindle microtubules in the sea urchin.
Orthokinetic and klinokinetic responses of human polymorphonuclear leucocytes
Authors: Keller H. U. , Zimmermann A.
Published: February 2005

Evidence is presented to show that klinokinesis, which was previously demonstrated in bacteria and amoeba only, may also occur in metazoan cells. The chemotactic peptide formyl‐Met‐Leu‐Phe (fMLP) elicited orthokinetic and klinokinetic responses of human blood‐borne polymorphonuclear leucocytes (PMNs) under the test conditions used. Increased speed (orthokinesis) was due to an increase in the proportion of migrating cells as well as in the speed of the locomoting subset. The klinokinetic effect was manifested by a decrease in the klinolocomotion index, the mean angle of changes in direction ⩾ 90°, and the frequency of turns ⩾ 90°. The klinolocomotion index was inversely related to speed. This explains the synergistic effect of klinokinesis and orthokinesis in this system. Colchicine alone had and orthokinetic effect which was exclusively due to alterations in the proportion of migrating cells and it altered the turning behaviour without exerting a klinokinetic effect. However, colchicine had marginal orthokinetic and klinokinetic effects on fMLP‐stimulated cells resulting in reduced translocation. The relationship between klinokinesis and mean angle or frequency of turns has been analysed. Klinokinesis was a substantial though not the major element of the chemokinetic response to fMLP under the conditions used. No other metazoan cells have been shown to possess such a complete pattern of responses, including orthokinesis, klinokinesis, and chemotaxis, which regulate locomotion.
A Ca2+ insensitive actin‐crosslinking protein from Dicytostelium discoideum
Authors: Brown Susan S.
Published: February 2005

We have isolated a 30,000‐dalton protein from Dictyostelium which cosedimented with and affected the low shear viscosity of actin. At low concentrations, this protein increased the low shear viscosity to greater than that of the actin control, whereas higher concentrations decreased viscosity. The viscosity decrease correlated with the formation of actin filament bundles, as seen electron microscopically. This protein resembled a previously reported actin‐binding protein from Dictyostelium [Fechheimer and Taylor, 84, J Biol Chem 259:4514] in electrophoretic mobility, Stokes radius, and ability to crosslink filaments, but was shown to be different by peptide mapping, lack of immunologic crossreactivity, and lack of sensitivity to calcium.
Alteration of the distribution of intermediate filaments in PtK1 cells by acrylamide II: Effect on the organization of cytoplasmic organelles
Authors: Eckert Barry S.
Published: February 2005

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.
The lack of interaction between vinculin and actin
Authors: Otto Joann J.
Published: February 2005

Vinculin was purified from chicken gizzard by a modification of the method of Feramisco and Burridge [1980; J Biol Chem 255:1194]. Vinculin did not alter the viscosity of actin as measured in an Ostwald viscometer, nor did it affect actin polymerization as measured with the fluorescent NBD‐actin assay. Sedimentation experiments demonstrated that vinculin did not bind to actin, and electron microscopy of negatively stained specimens indicated that vinculin did not aggregate actin filaments into bundles. These results suggest that vinculin, by itself, does not interact with actin at least under commonly used conditions to assay actin‐protein interactions in vitro.
A protein of 175,000 daltons associated with striated rootlets in ciliated epithelia, as revealed by a monoclonal antibody
Authors: Klotz Catherine , Bordes Nicole , Laine M. Christine , Sandoz Daniel , Bornens Michel
Published: February 2005

Basal bodies from laying quail oviduct were semipurified and used as immunogen to produce monoclonal antibodies. On 38 clones obtained and among those staining the apical pole of the ciliated cell, CC‐310 was chosen because it labeled the apical region with a punctated aspect, suggesting a staining of basal bodies or of basal body‐associated structures; the basal pole was also labeled.

The ultrastructural localization performed by the immunogold technique showed that the labeling was mainly associated with the striated rootlets. The basal feet, the side of the basal bodies, and the basal poles of the demembranated cells were also decorated. The identification of the antigen performed by immunoblots of deciliated cortices revealed two proteins of 175,000 and 40,000, whereas immunoblots of basal bodies showed only the 175,000‐mw protein. The possibility of these two proteins sharing the same epitope, located at both poles of the cell, is discussed.

Immunofluorescence ascertained that CC‐310 decorated the striated rootlets in ciliated epithelia from other species: mussel, frog, and human tissue. Finally, when tested on cultured cell lines, CC‐310 labeled the centrosome and its associated rootlets on PtK2 during interphase. During mitosis the poles of the mitotic spindle were stained without any apparent rootlet‐like structure.
The origin of flagella–autogenous or symbiontic?
Authors: Sleigh Michael A.
Published: February 2005

Earlier hypotheses of the origin of flagella appear untenable in the light of recent evidence on the ancestry of eukaryotes. It is suggested that microtubules and flagella evolved early in eukaryote evolution to enhance phagotrophy.
Patterns of flagellar wave propagation in Crithidia oncopelti at increased viscosities
Authors: Hirons M. R. , Holwill M. E. J.
Published: February 2005

In its normal culturing environment, the trypanosomid flagellate Crithidia oncopelti propagates basally‐directed planar waves, but may under certain conditions exhibit base‐to‐tip wave propagation in what is regarded as an avoidance response. The beat frequency and wave shape in both modes of beating are dependent on the viscosity of the swimming medium; viscosity may also influence the direction of wave propagation. If Crithidia experience a sudden increase in viscosity, there is a marked increase in the proportion of the population that is seen to exhibit wave propagation from base to tip; this proportion gradually decreases with time until the whole sample has reverted to “normal” beating. In a single organism, the resumption of normal beating is not accomplished in a single transition but by a series of switches between the forward and reverse modes. The interval of time between successive switches appears to be random, while the length of time spent in base‐to‐tip wave propagation gradually decreases. Despite the randomness of the switching process, its rate when averaged over successive time intervals is found to be constant at a particular viscosity and also dependent on it. The precise manner by which this organism is able to control its direction of wave propagation is unclear. However, the switching behavior it exhibits during the period of adaptation to an increased mechanical loading of the flagellum may reflect a process that characterizes a facet of this controlling mechanism.
Microtubule‐dependent reticulopodial motility: Is there a role for actin?
Authors: Travis Jeffrey L. , Bowser Samuel S.
Published: February 2005

We summarize our recent immunocytochemical characterization of the reticulopodial cytoskeleton of two allogromiid foraminifers and our pharmacologic dissection of its motility. The reticulopodial microtubule cytoskeleton stained with an antiserum to brain microtubule‐associated protein 2. Polymeric actin was localized in the reticulopodia by rhodamine‐phalloidin staining. Microtubule inhibitors reversibly inhibited all aspects of motility; cytochalasins induced altered morphology and disorganization of motility but did not inhibit pseudopodial movements or intracellular transport. Simultaneous application of KCN and salicylhydroxamic acid (an alternative oxidase inhibitor) rapidly blocked all movement, indicating that motility is dependent on metabolic energy and that an alternative oxidative pathway functions in allogromiids. Micromanipulation and laser microsurgical experiments revealed tension throughout the reticulopodium. Our results suggest that microtubules are active components of the reticulopodial motile machinery. Actin may mediate substrate adhesion, whole‐cell locomotion, pseudopodial tension, and coordination of the microtubule‐based motility.
In vitro movements of actin and myosin filaments from muscle
Authors: Higashi‐Fujime Sugie
Published: February 2005

Unidirectional sliding of myosin filaments along F‐actin bundles was produced with purified muscle actin and myosin in the presence of ATP. The velocity of myosin filament sliding was independent of myosin filament length. This result supports a recent hypothesis that long distance movement of myosin cross‐bridge can be induced by splitting of one ATP molecule [Yanagida, Arata, and Oosawa, 1985. Nature. 316:366–369; Higashi‐Fujime. 1985. J. Cell Biol., 101:2335–2344].
Cell surface displacement during euglenoid movement and its computer simulation
Authors: Suzaki Toshinobu , Williamson Richard E.
Published: February 2005

We recently showed by videomicroscopy that adjacent pellicular strips slide relative to each other without contraction during S‐shaped bending movement in Euglena fusca (Suzaki and Williamson, 1985). In order to validate this sliding strip mechanism for other species and other shape changes, a theoretical analysis and a computer simulation were carried out. Some of the commonly observed euglenoid cell shapes (rounded. S‐shaped, and embryo‐like shapes) were generated. Our results suggest that Euglena probably achieves a variety of cell shape changes by means of locally regulated sliding between adjacent pellicular strips of constant length and width.
Calcium‐modulated contractile proteins associated with the eucaryotic centrosome
Authors: Salisbury J. L. , Baron A. T. , Coling D. E. , Martindale V. E. , Sanders M. A.
Published: February 2005

Affinity‐purified antibodies that recognize the 20,000‐dalton molecular weight (20 kd) striated flagellar root protein of Tetraselmis striata have been used to identify antigenic homologs in other eucaryotic organisms of diverse evolutionary origins. Among the green algae, Tetraselmis and Chlamydomonas, and their colorless relative, Polytomella, the 20‐kd homologs appear associated with basal bodies. This occurs most prominnently in the form of flagellar roots of both striated and microtubule subtended types. Among cultured mammalian cells (PtK2 and primary mouse macrophage cell lines), flagellar root protein homologs appear as basal feet, pericentriolar fibrils, and pericentriolar satellites. Mammalian sperm cells also show flagellar root protein homologs associated with their basal bodies. We envisage a functional role for these fibrous calcium‐sensitive contractile proteins in altering the orientation of centrioles or basal bodies with their associated MTOCs by responding to topological calcium fluxes.
Motility mechanisms in the actinopods (Protozoa): A review with particular attention to axopodial contraction/extension, and movement of nonactin filament systems
Authors: Febvre‐Chevalier Colette , Febvre Jean
Published: February 2005

Several motility phenomena displayed by members of the Heliozoa, Radiolaria, and Acantharia (Protozoa, Actinopoda) are reviewed. These phenomena include (1) cytoplasmic streaming, internal saltatory motion, and transport of particles at the cell surface; (2) axopod contraction and extension; and (3) contraction of nonactin filament systems.

Cytoplasmic streaming and saltatory motion require energy derived from oxidative phosphorylation. In addition, calcium appears to be involved in the regulation of these movements, and a role for calmodulin is suggested. At present, the molecular basis for these motility phenomena remains obscure.

We have focused our attention on the rapid contraction of axopods and stalk in the marine heliozoan Actinocoryne contractilis (Febvre‐Chevalier: J. Marine Biol. Assoc. U.K. 60:901‐928, 1980). Contraction is accompanied by the cataclysmic breakdown of microtubules. For this species, a Na+ and Ca2+‐dependent action potential precedes axopod contraction. A lack of contraction in Ca2+‐free media (10−7 M Ca2+) suggests that Ca2+ fluxes across the cell membrane are required.

Motile phenomena associated with nonactin filaments of fibrous systems in actinopods, especially in the myonemes of the acantharians (Febvre and Febvre‐Chevalier: Biol. Cell. 44:283–304, 1982) are also examined. In vitro contraction of these filaments is CA2+ dependent and ATP independent' cycles of contraction and extension are caused by Ca2+‐dependent conformational changes in pairs of twisted filaments. In vivo, the Ca2+ dependent contraction of these myonemes may be independent of direct mitochondrial control, but metabolic ATP and calmodulin may be required to regulate the level of free cytoplasmic calcium.
The stabilization of microtubules in isolated spindles by tubulin‐colchicine complex
Authors: Hays T. S. , Salmon E. D.
Published: February 2005

We have analyzed the effect of colchicine and tubulin dimer‐colchicine complex (T‐C) on microtubule assembly in mitotic spindles. Cold‐ and calcium‐labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly‐disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a halftime of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 μM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 μM T‐C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T‐C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T‐C in vivo. These results support the current view that colchicine does not directly promote microlubule depolymerization. Rather, it is T‐C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation.

In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly. The rate of tubulin dissociation from spindle microtubules in vitro in reassembly buffer without soluble tubulin is about 20 times slower than the rate of dissociation in vivo when assembly is blocked abruptly by T‐C. The rate of tubulin dissociation from the spindle microtubules may determine their response to T‐C, since the tubulin dissociation rate in vivo is about 12 times faster than the rate measured here for spindle microtubules in standard microtubule reassembly buffer at physiological temperature.