bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of vanadate on the assembly and disassembly of purified tubulin
Authors: Kirazov Evgeni P. , Weiss Dieter G.
Published: February 2005

Sodium‐orthovanadate (100–700 μM) added to purified pig brain microtubule protein (molar ratios 13–90 moles vanadate/mole tubulin) inhibits to a considerable extent the assembly (up to 65%) and the disassembly rates (up to 60%) of microtubules, as determined by turbidimetry. Vanadate added to preformed microtubules did not appreciably alter the turbidity level of the samples, however, the disassembly rates were decreased in the same manner as when vanadate was added prior to polymerization. Microtubule protein kept on ice for 3–6 hours became more susceptible to vanadate than freshly prepared protein. The effect of vanadate was independent of the GTP concentration at which the polymerization assays were performed (0.025 to 1 mM GTP). In the presence of taxol, which increases the rate and extent of microtubule formation, vanadate had no effect on assembly rates. Disassembly was inhibited, however, much less than in the presence of vanadate alone. Electron microscopy and polyacrylamide gel electrophoresis did not reveal differences between microtubules prepared in the presence or in the absence of vanadate. This is consistent with the notion that vanadate does not interfere with the interaction between tubulin and the high‐molecular weight microtubule‐associated proteins. Apparently vanadate brings about an allosteric change of the microtubule protein(s) resulting in the abnormal polymerization kinetics of tubulin found in our study. The above results may be relevant for studies where the effects of vanadate on intracellular motility are interpreted as being solely due to a specific inhibition of ATPases.
Computer simulation of bend propagation by axoplasmic microtubules
Authors: Brokaw Charles J.
Published: February 2005

The generation of bending waves by microtubules in squid nerve axoplasm has been modelled using appropriately modified versions of computer programs developed previously for simulation of flagellar bending waves. The results confirm that a constant longitudinal force directed along the axis of the microtubule is sufficient to cause the generation of regular oscillations and propagated bending waves when the forward gliding movement of the microtubule is obstructed. No control mechanism is required to modulate the active force‐generating system. In order to obtain bending waves similar to those observed experimentally, it was necessary to use a model for the force‐generating system in which the active force decreases with increasing sliding velocity. If the elastic bending resistance of axoplasmic microtubules is similar to that of microtubules in sperm terminal filaments, the longitudinal force per unit length generated by the axoplasmic microtubules must be of the same order of magnitude as the force generated by dynein arms along the doublet microtubules of eukaryotic flagella.
Malorientation and abnormal segregation of chromosomes during recovery from Colcemid and Nocodazole
Authors: Ladrach Kenneth S. , LaFountain James R.
Published: February 2005

Reversal of meiotic arrest in crane‐fly spermatocytes by U.V. irradiation of Colcemid‐arrested cells or by rinsing Nocodazole‐arrested cells in fresh buffer results in the induction of chromosome malorientation. Malorientations observed among Colcemid‐recovering and Nocodazole‐recovering spermatocytes at frequencies higher than normally observed in untreated cells included associations of sister kinetochores of half‐bivalents with both spindle poles (amphitely), in contrast with associations of sisters with only one pole (syntely) as is usually found during the first meiotic division. In several cases, prior to anaphase onset, maloriented bivalents appeared unusually tilted with respect to the spindle axis, and during anaphase they gave rise to laggard half‐bivalents that did not segregate during anaphase along with half‐bivalents having proper syntelic orientation. The results parallel previous findings obtained during cold recovery, and the properties of the drugs used here suggest that their action on microtubules, although reversible, induces malorientation during recovery from meiotic arrest.
Kinetochore microtubules in crane‐fly spermatocytes: Untreated, 2°C‐treated and 6°C‐grown spindles
Authors: Scarcello Lisa A. , Janicke Marie A. , LaFountain James R.
Published: February 2005

We investigated the involvement of kinetochore microtubules (kMTs) in mediating chromosome‐to‐pole connections in crane‐fly (Nephrotoma suturalis and Nephrotoma ferruginea) spermatocytes. Two experimental treatments were used to yield spindles with reduced numbers of nonkinetochore microtubules (nkMTs). Short‐term (10–15 min) exposure of spermatocytes to 2°C caused depolymerization of the majority of nkMTs, resulting in a kMT:(kMT + nkMT) ratio of 0.76. Long‐term (24h) exposure to 2°C followed by recovery at 6°C resulted in a kMT:(kMT + nkMT) ratio of 0.55, the spindle having more nkMTs than a 2°C‐treated spindle but fewer than an untreated spindle, in which the kMT:(kMT + nkMT) ratio was 0.27. The numbers and lengths of kMTs in 6°C‐grown spindles were similar to those in untreated cells, suggesting that the overall inhibition of MT assembly at 6°C apparently did not affect the mechanism by which kMTs are formed. We observed most kMTs of early anaphase spindles to be long (>3 μm), and many extended to the polar regions of the spindle. Thus, the crane‐fly spindle appears not to be as atypical as it was previously suggested to be.
Dynamic aspects of the contractile system in Physarum Plasmodium: I. Changes in spatial organization of the cytoplasmic fibrils according to the contraction‐relaxation cycle
Authors: Ishigami Mitsuo
Published: February 2005

Dynamic changes in the spatial organizations of cytoplasmic fibrils (microfilament bundles) related to the contraction‐relaxation cycle in thin‐spread plasmodia of Physarum polycephalum were investigated by fluorescence microscopy, where NBD‐phallacidin was used to stain the fibrils, combined with polarizing light microscopy.

The fibrillar organization in the anterior region, which consists of a fanlike spreading plasmodial sheet, strikingly changed according to the phase of the cycle. In the early stage of the contraction, as the endoplasm began to stream backward, the fibrils developed into a number of slender and flabby fibrils emanating from the inside of the cell membrane and the nodes. They became thicker and more straightforward fibrils running parallel to each other at the middle stage, and finally formed a thick framework consisting of a “polygonal network” near the tip of the migrating front and a “parallel array” in the inner part. In the relaxation phase, as the endoplasm streamed forward, the fibrillar framework disintegrated gradually and finally disappeared almost completely, remaining only around the nodes in some cases.

The fibrillar patterns in the posterior region, which consists of ramified strands, showed no conspicuous rhythmic change with alternation of the streaming direction.
Cytoskeletal architecture of reactivated crayfish axons, with special reference to crossbridges among microtubules and between microtubules and membrane organelles
Authors: Hirokawa Nobutaka , Yorifuji Hiroshi
Published: February 2005

Bidirectional organelle movements were observed in fresh and permeabilizedreactivated (0.02% saponin, 5 mM Mg++ ATP) walking leg axons of crayfish with video‐enhanced contrast, differential interference contrast (AVEC‐DIC) microscopy; and the cytoskeletal organization of those axons was studied with quickfreeze, deep‐etch electron microscopy (QF,DE) to understand the structure of the microtubule (MT) domain and to determine the basic cytoskeletal structures necessary for organelle transport in vivo. Vesicles and mitochondria moved bidirectionally in the central parts of fresh or permeabilized‐reactivated axons. Although the axoplasm of the fresh axon was composed of longitudinally oriented microtubules and granular materials in which membrane organelles were embedded, a network of fine strands existed in the core of the granular materials. Crossbridges between membrane organelles and microtubules were present. In the central part of reactivated axons, the cytoskeleton consisted of microtubules, highly anastomosing networks of fine strands (6.6 ± 1.4 nm in width) that crosslinked the microtubules with each other, and relatively short, straight crossbridges (25 ± 3.9 nm in length, 5.5 ± 2.1 nm in width) crosslinking membrane organelles with microtubules. It has been shown that a 270KD microtubule associated protein (MAP) could be a main component of crossbridges between MTs [Hirokawa, 1986]. Hence the dynamic conformational change of crossbridges between membrane organelles and microtubules could play an important role when membrane organelles are transported.
Effects of taxol on microtubule arrays in cultured higher plant cells
Authors: Weerdenburg Carol , Falconer Marcia M. , Setterfield George , Seagull Robert W.
Published: February 2005

Treatment with 10 μm taxol disrupted mitotic and cytoplasmic arrays of microtubules (MT) in cultured cells of two higher plants, Vicia hajastana (vetch) and Zinnia elegans. When treated for 1, 24, and 48 h, cells in both cultures showed similar effects. After 1 h, multipolar arrays of MT were noted in prophase, large aster‐like arrays of MT appeared in metaphase, and extra MT shared poles with otherwise normal‐appearing metaphase and anaphase configurations. After 24 and 48 h, some phragmoplasts were multipartite or misplaced. In interphase cells, micronuclei and multinucleate cells were evidence of irregular mitosis and cytokinesis. Cytoplasmic MT in elongated cells were oriented parallel to, instead of at right angles to the long axis of the cell. Some interphase cells lost asymmetry while maintaining organized arrays of MT. Taxol appears to disrupt mitotic and cytoplasmic arrays of MT, seemingly overriding the mechanism(s) regulating MT polymerization and orientation.
Video analysis of chemotactic locomotion of stored human polymorphonuclear leukocytes
Authors: Burton Jarrett L. , Law Ping , Bank Harvey L.
Published: February 2005

Previous studies of the storage of polymorphonuclear leukocytes (PMNs) have used an empirical approach to define “optimal” conditions. To date, no storage conditions have been described which satisfactorily preserve the chemotactic function of PMNs beyond 24 h. In an effort to define the precise nature of the storage lesion, we studied the chemotactic locomotion of freshly isolated PMNs and PMNs which had been suspended in citrate‐phosphate‐dextrose‐adenine (CPD‐Al) plasma and stored in PVC bags, at 20–22°C for 24 h. We used time‐lapse video recording and computer image analysis to quantitate the motion of PMNs migrating under agarose. The positions of individual motile cells were traced at 1‐min intervals for 5 min. The following parameters were used to quantitate migration: (1) speed (distance/min), (2)) persistence of locomotion index (velocity/speed), (3) orientation angle (the angle of the vector describing the next displacement of a cell relative lo a direct line toward the chemoattractant), and (4) chemotropic index (cosine of the orientation angle). After 24 h of storage, the following changes were observed: (1) fewer cells migrated, (2) (he speed of migrating cells was reduced by 25%, (3) the persistence of locomotion index decreased by 7%, which indicates that migrating cells made slightly more/wider turns, and (4) the chemotropic index was decreased by 30%, which indicates that migrating cells were less accurate in their orientation toward the chemoattractant. Apparently, the storage of PMNs selectively impairs the ability of some cells to orient accurately in a chemotactic gradient and changes the distribution of these locomotor parameters within the population.
Protein‐protein interactions in the 18S ATPase of Chlamydomonas outer dynein arms
Authors: Mitchell David R. , Rosenbaum Joel L.
Published: February 2005

When outer‐row dynein arms are extracted from Chlamydomonas flagellar axonemes, they dissociate into two ATPase complexes with sedimentation coefficients of 12S and 18S. We immunized mice with 18S dynein and generated a library of monoclonal antibodies against the polypeptides in this complex. Antibodies were selected which specifically recognize the 18S α‐ and β‐heavy chains and the 83,000‐dalton and 70,000‐dalton intermediate chains. These antibodies were isolated and characterized for their ability to recognize determinants on both denatured antigens and native 18S dynein; 18S dynein was dissociated in stepwise fashion into smaller aggregates with ionic and nonionic detergents and the resulting subcomplexes were isolated by precipitation with specific monoclonal antibodies. The smallest aggregates isolated were heterodimers between the α‐chain and a 16,000‐dalton light chain and between the two intermediate chains. Additional close associations of the β‐heavy chain with an 18,000‐dalton light chain and 70,000‐dalton intermediate chain, and a weaker interaction between the intermediate chain heterodimer and light chains of 21,000 daltons and 12,500 daltons, were also observed. We present a model of 18S dynein substructure based upon this information.
Comparison of the beating of cis‐ and trans‐flagella of Chlamydomonas cells held on micropipettes
Authors: Rüffer Ursula , Nultsch Wilhelm
Published: February 2005

Chlamydomonas cells sucked onto micropipettes were filmed at 500 frames/sec and analyzed as to their forward beating mode. A comparison with freely swimming cells revealed that the flagella of the sucked cells beat in a normal threedimensional manner, with beat frequencies that correspond to those of freely swimming cells. Most beats were synchronous. but not symmetrical; cis‐ and trans‐flagellum appear to beat in a slightly different manner. Some cells beat synchronously throughout, but mostly synchrony was interrupted by a single asynchrony or up to incessant asynchronies, caused by transient accelerations of the trans‐ (fo‐) flagellum. Only rarely did cis‐ and trans‐flagella have different but constant beat frequencies. Helical swimming of Chlamydomonas more likely is due to the beat asymmetries of the two flagella than to differences of beat frequencies. In our records, the stigma is on the inside of the helical swimming path.
A unique tubulin antibody which disrupts particle movement in squid axoplasm
Authors: Johnston Karen M. , Brady Scott T. , van der Kooy Derek , Connolly Joe A.
Published: February 2005

Microtubules have been demonstrated to be a substrate for organelle transport and particle translocation in vitro and in vivo. Subsequent to a previous report of inhibition of axonal transport of exogenous tracers in vivo using antiserum NS‐20 against tubulin (Johnston et al: Brain Res. 1986), we now show disruption of particle movement in extruded squid axoplnsm using this unique immunological probe. Using video‐enhanced contract‐differential interference contrast (AVEC‐DIC) microscopy, we examined the properties of particle movement along microtubules and demonstrated that bolh the velocity of particle movement and the numbers of particles moving are decreased in the presence of NS‐20 antiserum or NS‐20 affinity‐purified antibodies but. not in the presence of another antiserum against tubulin. The amount of microtubule substrate does not change in the presence of any of the antisera. In conclusion, we suggest that NS‐20 antibodies bind near or at a site on the tubulin molecule which is critical in the mechanism of particle transport, and provide a direct immunological probe to examine the mechanism of microtubule involvement in axonal transport.
cAMP‐mediated inhibitory effect of calmodulin antagonists on ciliary reversal of Paramecium
Authors: Izumi Akemi , Nakaoka Yasuo
Published: February 2005

To explore possible roles of calmodulin in Ca2+‐induced ciliary reversal, we tested the effects of calmodulin antagonists on Triton‐extracted models of Paramecium. In the extracted models prepared by the method of Naitoh and Kaneko [Science 176:523–524, 1972], the Ca2+ ‐induced ciliary reversal was not inhibited by calmodulin antagonists, trifluoperazine (TFP), or 5‐chloro‐l‐naphthalenesulphone amide (W‐7). However, in the presence of adenosine 3′,5′‐cyclic mono‐phosphate (cAMP), whose concentration is below the one that alters the ciliary direction, TFP inhibited ciliary reversal and the models swam forward at 10−5 M Ca2+. When the washing medium in the preparation of the extracted models was replaced with one containing MgCl2, the extracted model showed sensitivity to calmodulin antagonists without addition of cAMP; at 10−5 M Ca2+, 40 μM TFP or 100 μM W‐7 inhibited the ciliary reversal and the models swam forward. Such effect of antagonists was abolished by an inhibitor of cAMP‐dependent protein kinase. On the other hand, addition of cAMP enhanced the inhibitory effect of antagonists. These results suggest that calmodulin antagonists act to increase the extent of cAMP‐dependent protein phosphorylation that inhibits the Ca2+ ‐induced ciliary reversal.
Primary cilia cycle in PtK1 cells: Effects of colcemid and taxol on cilia formation and resorption
Authors: Jensen Cynthia G. , Davison Edwin A. , Bowser Samuel S. , Rieder Conly L.
Published: February 2005

The effects of colcemid (0.16–1.0 μM) and taxol (10 μM) on the primary cilia cycle in PtK1 cells were studied by antitubulin immunofluorescence microscopy and by high‐voltage electron microscopy of serial 0.25‐μm sections. Although these dings induce a fully characteristic rearrangement (taxol) or disassembly (colcemid) of cytoplasmic microtubulcs, neither affects the structure of primary cilia formed prior to the treatment or the resorption of primary cilia during the initial stages of mitosis. Cells arrested in mitosis by taxol or colcemid remain in mitosis for 5–7 h at 37°C and then form 4N “micronucleated” restitution nuclei. Formation of primary cilia in these micronucleated cells is blocked by colcemid in a concentration‐dependent fashion: normal cilia with expanded (ie, bulbed) distal ends form at the lower (0.16–0.25 μM) concentrations, while both cilia formation and centriole replication are inhibited at the higher (≥ 1.0 μM) concentrations. However, even in the presence of 1.0 μM colcemid, existing centrioles acquire the appendages characteristically associated with ciliating centrioles and attach to the dorsal cell surface. Continuous treatment with colcemid thus produces a population of cells enriched for the early stages of primary cilia formation. Micronucleated cells formed from a continuous taxol treatment contain two normal centriole pairs, and one or both parenting centrioles possess a primary cilium. Taxol, which has been reported to stabilize microtubules in vitro, does not inhibit the cell‐cycle–dependent assembly and disassembly of axonemal microtubules in vivo.
Effect of inhibition of the catalytic activity of cyclic amp‐dependent protein kinase on mitosis in PtK1 cells
Authors: Browne Carole L. , Bird Mary Lynn , Bower William
Published: February 2005

Evidence has suggested that cyclic AMP, acting through activation of the type II cyclic AMP‐dependent protein kinase, may play a role in the regulation of interphase and mitotic microtubules. In order to examine the potential role of the type II cAMP‐dependent kinase during mitosis, dividing PtK1 cells were microinjected with two specific inhibitors of the catalytic activity of the type II kinase. These inhibitors were a specific protein inhibitor of cAMP‐dependent protein kinase (PKI) and an affinity‐purified polyclonal antiserum (anti‐C) directed against the catalytic subunit of the kinase. Both have been shown previously to inhibit kinase activity in vitro. Microinjection of PKI during early‐ to mid‐prophase significantly delayed the progression of the cells through mitosis, with the greatest delay occurring in metaphase. PKI injected during prometaphase also delayed progression through mitosis but to a lesser extent. Microinjection of anti‐C during early‐ to mid‐prophase also caused a significant delay in the completion of mitosis, with many cells becoming “hung up” in prometaphase. Anti‐C injected during prometaphase had little effect on subsequent progression through mitosis. Microinjection of either anti‐C or PKI during metaphase had no discernible effect. No effect on anaphase movement of chromosomes was observed with any treatment. These results provide further evidence that cAMP‐dependent phosphorylation may be involved in the regulation of mitosis, although whether it acts directly through regulation of mitotic spindle microtubules is unclear.
Cytochalasin B‐induced redistribution of cytokeratin filaments in PtK1 cells
Authors: Wolf Kathryn M. , Mullins J. Michael
Published: February 2005

Indirect immunofluorescence demonstrated a dramatic reorganization of cytokeratin filaments produced by cytochalasin B (CB) treatment of PtK1 cells. Much of the normal cytokeratin network became arranged into a latticework consisting of bundles of cytokeratin filaments that radiated from, and interconnected, distinct foci, Electron microscopy showed foci to be dense granular regions through which bundles of cytokeratin filaments looped. Composition of the foci included actin, myosin, and alpha‐actinin, as shown by labeling with rhodamine phalloidin or specific antisera. Simultaneous treatment with CB and colchicine was not required for lattice formation, but did produce more extensive development than did CB alone. In cells treated only with CB, the microtubule network remained intact, even in regions of extensive lattice formation. These results contrast sharply with those of Knapp et al (J. Cell Biol. 97:1788 [1983b]), who found lattice formation dependent upon simultaneous CB and colchicine treatment. Time‐course and dose‐response studies of CB treatment showed lattice formation to follow disruption of stress fibers and the concentration of actin into distinct patches that marked the location of lattice foci. Overall results suggest a structural association between microfilaments and cytokeratin filaments that produces the lattice pattern upon CB‐induced disruption of stress fibers. Lattice formation was not limited to a specific cell‐cycle stage, since G1, G2, and M cells displayed the lattice. Treatment of cells with dihydro‐CB and experiments with enucleated cells showed that lattice formation was dependent upon neither the inhibition of sugar transport nor the nuclear extrusion effects of CB.
Amebae of Dictyostelium discoideum respond to an increasing temporal gradient of the chemoattractant cAMP with a reduced frequency of turning: Evidence for a temporal mechanism in ameboid chemotaxis
Authors: Varnum‐Finney Barbara , Edwards Kevin B. , Voss Edward , Soll David R.
Published: February 2005

In an aggregation territory of Dictyostelium discoideum, outwardly moving, nondissipating waves of the chemoattractant cAMP sweep across each ameba. At the front of each wave, an ameba experiences an increasing temporal and a positive spatial gradient of cAMP. At the back of a wave, an ameba experiences a decreasing temporal and a negative spatial gradient of cAMP. Employing a perfusion chamber, we have mimicked the temporal dynamics of these waves in the absence of a spatial gradient and demonstrated that the frequency of lateral pseudopod formation and the frequency of turning are dramatically affected by the direction and dynamics of the temporal gradient. In addition, since an ameba will move in a directed fashion up a shallow, nonpulsatile gradient of cAMP, we also mimicked the increasing temporal gradient generated by an ameba moving up a shallow spatial gradient. The frequency of lateral pseudopod formation and the frequency of turning were depressed. Together, these results demonstrate that amebae can assess the direction of a temporal gradient of chemoattractant in the absence of a spatial gradient and alter both the frequency of pseudopod extension and turning, accordingly. Although these results do not rule out the involvement of a spatial mechanism in assessing a spatial gradient, they strongly suggest that the temporal dynamics of a cAMP wave or the temporal gradient generated by an ameba moving through a spatial gradient may play a major role in chemotaxis.
Endoplasmic microtubules connect the advancing nucleus to the tip of legume root hairs, but F‐actin is involved in basipetal migration
Authors: Lloyd Clive W. , Pearce Karen J. , Rawlins David J. , Ridge Robert W. , Shaw Peter J.
Published: February 2005

A prominent feature of tip growth in filamentous plant cells is that the nucleus often migrates in step with the tip as it extends. We have studied this long‐recognized but unexplained relationship in root hairs of the legume Vicia hirsuta by a variety of microscopic techniques. Using rhodaminyl lysine phallotoxin, and antitubulin antibodies, root hairs are shown to contain axial bundles of F‐actin and a complex microtubular system. To the basal side of the nucleus the microtubules are cortical and net axial but in the region between nucleus and tip the arrangement is more complicated. Electron microscopic thin sections demonstrate that internal bundles of microtubles exist in addition to the plasma membrane‐associated kind. Computerized deblurring of through‐focal series of antitubulin stained hairs clarifies the three‐dimensional organization: bundles of endoplasmic microtubules progress from the nuclear region toward the apical dome where they can be seen to fountain out upon the cortex.

The relationship between nucleus and tip can be uncoupled with antimicrotubule herbicides. Time lapse video microscopy shows that these agents cause the nucleus to migrate toward the base. This contrary migration can be inhibited by adding cytochalasin D, which fragments the F‐actin bundles.

It is concluded that microtubules connect the nucleus to the tip but that F‐actin is involved in basipetal migration as is known to occur when symbiotic bacteria uncouple the nucleus from the tip.
Platelet intermediate filaments: Detection of a vimentinlike protein in human and bovine platelets
Authors: Tablin Fern , Taube David
Published: February 2005

Human and bovine platelets contain a 58,000‐dalton vimentinlike protein that cross‐reacts with antivimentin antibody. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots indicate that this protein is present in whole platelet lysates and triton insoluble cytoskeletons. Transmission electron microscopy of platelets reveals an isotropic network of individual intermediate filaments distributed throughout the platelets. High salt, triton extracted, glutaraldehyde and tannic acid fixed platelets reveal 10‐nm filaments that can be seen to form a peripheral ring, as well as an isotropic network in the body of the cells. Indirect immunofluorescence of resting and spread platelets demonstrates a circumferential staining pattern close to the cell membrane, with additional fibrillar staining throughout the platelets. Our data suggest that the 58,000‐dalton vimentinlike protein may be associated with the microtuble coil and the plasma membrane, and may thus help to maintain the resting platelet's discoid shape.
Reorganization of microtubules during mitosis in Dictyostelium: Dissociation from MTOC and selective assembly/disassembly in situ
Authors: Kitanishi‐Yumura Toshiko , Fukui Yoshio
Published: February 2005

We applied the “agar‐overaly” immunofluorescence techinque (Yumura, S., H. Mori, and Y. Fukui, J. Cell Biol. 99:894–899, 1984) to a semisynchronous culture of Dictyostelium discoideum for studying the organization changes in the microtubule system during mitosis. Using a flurescent DNA dye DAPI (4′,6′ ‐diamidino‐2‐phenylindole), chromatin fibers and individual chromosomes were visible in cells prepared by this method, whereby the mitotic phase could be critically evaluated.

We found that a rapid shortening of the cytoplasmic microtubules was preceded by a structural dislocation from their organizing centers (MTOCs) in the midprophase, resulting in the transient occurrence of free microtubules in the cytoplasm. Statistic analyses showed that microtubule disassembly in prophase was diphasic. Initially long, wavy microtubules shortened from their distal ends. Following dissociation of their proximal ends from the MTOC, all microtubules initiated rapid disassembly, probably from both ends. During this process, microtubule assembly from the now duplicated spindle pole body (SPB) resumed.

This study also revealed novel information on the dynamics of the Dictyostelium mitotic spindle: 1) Half spindles interdigitate in the spindle center, and the extent of interdigition increases coincidentally with the spindle elongation, and 2) during the anaphase to telophase, a subpopulation of spindle microtubules elongates while the rest of the microtubules disasemble very rapidly.

Overall this study indicates the presence of elaborate mechanisms responsible for the selective assembly/disassembly of particular microtubule subpopulations in situ.
Subcortical rotation in Xenopus eggs: A preliminary study of its mechanochemical basis
Authors: Vincent Jean‐Paul , Scharf Stanley R. , Gerhart John C.
Published: February 2005

The amphibian egg undergoes a 30° rotation of its subcortical contents relative to its surface during the first cell cycle, a displacement of 350 μm in 50 min. This is directly visualized by following the movement of an array of Nile blue (a subcortical stain) spots applied to the egg periphery (Vincent, Oster, and Gerhart: Dev Bio 113:484–500, '86). We have investigated the mechanochemical basis of this unusual cell motility. Subcortical rotation depends on microtubule integrity during its entire course and is insensitive to inhibitors of microfilament assembly. It does not depend on newly synthesized proteins for its operation or timing, and it does not involve calcium‐dependent processes. Finally, we show that vegetal fragments of the egg can complete rotation on their own, indicating that mechanochemical components can operate locally in this hemisphere.
A critical role for the polarization of membrane recycling in cell motility
Authors: Kupfer Abraham , Kronebusch Paul J. , Rose John K. , Singer S. J.
Published: February 2005

This paper is concerned with the proposition that the insertion of membrane mass into the leading edge of a motile cell plays a critical role in directed cell migration. We show by immunofluorescence, with cells transfected with a cloned cDNA encoding the G‐protein of a temperature‐sensitive mutant of vesicular stomatitis virus, that the first cell surface appearance of the G‐protein is indeed at the leading edge of the motile cell. Two drugs capable of inhibiting directed cell migration, cytochalasin D and monensin, appear to function independently, the former by affecting the actin cytoskeleton without affecting the polarized insertion of membrane mass into the cell surface and the latter by abrogating membrane mass insertion without affecting the actin cytoskeleton.
Monoclonal antibodies against plant proteins recognise animal intermediate filaments
Authors: Parke J. M. , Miller C. C. J. , Cowell I. , Dodson A. , Dowding A. , Downes M. , Duckett J. G. , Anderton B. J.
Published: February 2005

Four monoclonal antibodies were raised against polypetides present in a highsalt detergent‐insoluble fraction from cells of Chlamydomonas reinhardtii. Indirect immunofluorescence microscopy of fibroblasts and epithelial cells grown in culture using these plant antibodies revealed staining arrays identical to those obtained with well characterised antibodies to animal intermediate filaments. Immunoflurescence microscopy of Chlamydomonas with these monoclonal antibodies and a monoclonal antibody that recognises all animal intermediate filaments (anti‐IFA) gave a diffuse, patchy cytoplasmic staining pattern. Both the plant antibodies and anti‐IFA stained interphase onion root tip cells in a diffuse perinuclear pattern. In metaphase through to telophase, the labelling patterns colocalised with those of microtubules. Labelling of the phragmoplast was also detected but not staining of the preprophase band. On Western blots of various animal cell lines and tissues, all the antibodies labelled known intermediate filament proteins. On Western blots of whole Chlamydomonas proteins, all the antiboides labelled a broad band in the 57,000 Mr range, and three antibodies labelled bands around 66,000 and 140,000 Mr but with varibale intensites. On Western blots of whole onion root tip proteins, all the antibodies labelled 50,0000 Mr (two to three bands) polypetides and a diffuse and around 60,000 Mr and three of the antibodies also labelled several polypeptides in the 90,000–200,000 Mr range. The consistent labelling of these different bands by several different monoclonal antibodies recognising animal intermediate filaments makes these polypetides putative plant intermediate filament proteins.
Regulation of activation state and flagellar wave form in epididymal rat sperm: Evidence for the involvement of both CA2+ and cAMP
Authors: Lindemann Charles B. , Goltz Jason S. , Kanous Kathleen S.
Published: February 2005

Rat sperm from the cauda epididymis exhibit increased motility, longevity, and a distinct circular pattern of flagellar curvature in response to 5 mM procaine‐HCI or 0.1 mM 8‐(N, N‐diethylamino)‐octyl‐3,4,5‐trimethoxybenzoate (TMB‐8), reagents that are thought to play a role in the immobilization of free cellular calcium. Triton X‐100‐extracted sperm models will exhibit the same pattern of motility and curvature as procaine‐ or TMB‐8‐activated cells, but only when calcium is removed by a strong chelating agent, and in the pesence of cAMP (3 μM). Demembranated sperm models produced from epididymal rat sperm are quiescent unless cAMP is added. In these sperm models, the presence or absence of free calcium mediates a transition in flagellar curvature. The increased activity of the procaine‐treated intact cells was not accompained by a change in cellular ATP content, nor was ATP availability the limiting factor in the quiescent sperm. Therefore, the increased motility produced by procaine is probably mediated by a fall in free intracellular Ca2+ accompained by a rise in cAMP. Our finding that calcium controls the curvature of sperm flagella may explain altered patterns of flagellar beating, such as the hyperactivated motility that sperm exhibit in the female reproductive tract.
In vitro effects of benzodiazepines on ciliogenesis in the quail oviduct
Authors: Boisvieux‐Ulrich Emmanuelle , Laine Marie‐Christine , Sandoz Daniel
Published: February 2005

Immature oviduct implants from quails stimulated by estrogen to induce ciliogenesis were submitted to the in vitro action of benzodiazepines in organotypic culture. Diazepam and medazepam were added to the culture medium for 24 or 48 hours and tissues were examined by transmission and scanning electron microscopy for alterations in ciliary differentiation.

Ciliogenesis was inhibited by both diazepam and medazepam, which affected mainly the migration of the basal bodies. Assembly of basal bodies was achieved normally in the cytoplasm, but their separation from generative complexes and migration toward the apical membrane were prevented. They remained in clusters around a deuteosome or eventually anchored to the close lateral plasma membrane.

Furthermore, the drugs affected mature beating cilia, which then appeared lying tangentially to the cell surface. Relation between basal bodies and cortical cytoskeleton seemed to be altered by the drugs, which implies that the bearing of cilia and probably the ciliary beating movement were modified. Mocrovillus development was also altered by the action of these drugs.
Identification and characterizaton of a protein associated with the stembody using autoimmune sera from patients with systemic sclerosis
Authors: Kingwell B. , Fritzler M. J. , Decoteau J. , Rattner J. B.
Published: February 2005

An autoantibody that binds an antigen localized to the stembody of dividing cells has been identified in a patient with systemic sclerosis. Initially, this antigen is associated with the surface of the metaphase chromosomes. At the onset of anaphase the antigen becomes preferentially associated with the forming stembodies. This association is maintained as furrowing progresses during telophase and continues after the intercellular bridge is released from the daughter cells during G‐1. Immunoblots indicate that the epitope detected by immunoflurorescence is present on a protein with an apparent molecular weight of 38 kD.
Dynamic behavior of the transferrin receptor followed in living epidermoid carcinoma (A431) cells with nanovid microscopy
Authors: De Brabander M. , Nuydens R. , Geerts H. , Hopkins C. R.
Published: February 2005

Transferrin receptors labeled with the B3/25 monoclonal antibody–gold complexes were followed in living A431 cells by using video‐enhanced contrast microscopy. Initially, the antibody‐gold complexes bind to receptors which are freely mobile on the upper cell surface; they then become trapped at the inner margins of the peripheral lamellae and internalize. During endocytosis discrete gold‐loaded vesicular elements first appear, and then, as they fuse, a heterogenous peripheral endosomal compartment forms. The endosomes from this compartment then begin to migrate centripetally through the cytoplasm in a saltatory way so that within 15 min gold label accumulates in a juxtanuclear endosome compartment. This compartment, which consists mainly of multivesicular bodies, is thus formed by the influx and retention of peripheral endosomal elements and their continued fusion in the juxtanuclear area. Although their overall migration is inward, saltating endosomes frequently reverse their direction of movement. As label builds up in the juxtanuclear area, small vesicles containing gold label continuously pinch off from the larger elements and migrate toward the cell periphery.

Experiments with nocodazole and sodium azide show that the saltatory movements, the accumulation and retention of endosomes in the juxtanuclear area, and the separation of vesicles from endosomes are driven by a microtubule‐associated, ATP‐dependent, motility‐generating mechanism.

Analysis of the movements shows that although each individual vesicle saltation can occur unpredictably toward the centre or the periphery of the cell, a net centripetal flux is observed. Moreover, it is evident that the probability of migration toward and maintenance in the juxtanuclear area is related to the diameter of the vesicles. We propose a mechanism by which bidirectional saltation along microtubules forming a radial network may be instrumental in the selective concentration of large endosomes in the juxtanuclear area while small vesicles are left free to return to the periphery. This process may be responsible for the sorting of receptors and ligands destined either for intracellular degradation in juxtanuclear lysosomes or, alternatively, for recycling to the plasma membrane.
Ultrastructure and motion analysis of permeabilized paramecium capable of motility and regulation of motility
Authors: Lieberman Steven J. , Hamasaki Toshikazu , Satir Peter
Published: February 2005

Structural and behavioral features of intact and permeabilized Paramecium tetraurelia have been defined as a basis for study of Ca2+ control of ciliary reversal. Motion analysis of living paramecia shows that all the cells in a population swim forward with gently curving spirals at speeds averaging 369 ± 19 μm/second. Ciliary reversal occurs in 10% of the cell population per second. Living paramecia, quick‐fixed for scanning electron microscopy (SEM), show metachronal waves and an effective stroke obliquely toward the posterior end of the cell. Upon treatment with Triton X‐100, swimming ceases and both scanning and transmission electron microscopy reveal cilia that uniformly project perpendicularly from the cell surface. Thin sections of these cells indicate that the ciliary, cell, and outer alveolar membranes are greatly disrupted or entirely missing and that the cytoplasm is also disrupted. These permeabilized paramecia can be reactivated and are capable of motility and regulation of motility. Motion analysis of cells reactivated with Mg2+ and ATP in low Ca2+ buffer (pCa7) shows that 71% swim forward in straight or curved paths at speeds averaging 221 ± 20 μm/second. When these cells are quick‐fixed for SEM the metachronal wave patterns of living, forward swimming cells reappear. Motion analysis of permeabilized cells reactivated in high Ca2+ buffers (pCa 5.5) shows that 94% swim backward in tight spirals at a velocity averaging 156 ± 7 μm/second. SEM reveals a metachronal wave pattern with an effective stroke toward the anterior region. Although the permeabilized cells do not reverse spontaneously, the pCa response is preserved and the Ca2+ switch remains intact. The ciliary axonemes are largely exposed to the external environment. Therefore, the behavioral responses of these permeabilized cells depend on interaction of Ca2+ with molecules that remain bound to the axonemes throughout the extraction and reactivation procedures.
Membrane IgM: Interactions with the cortical cytoskeleton in the human lymphoblastoid cell line WiL2
Authors: Salisbury J. L. , Baron A. T. , Keller G. A. , Skiest D.
Published: February 2005

Cell‐surface IgM (antigen receptor) sediments with the membrane fraction following osmotic lysis and homogenization of cells of the human lymphoblastoid cell line WiL2. In nonreducing buffers, SDS PAGE analysis of membrane pellets demonstrates that “native” membrane IgM exists as a dimer. In contrast to osmotic lysis, lysis of cells with the nonionic detergent Triton X‐100 releases approximately 90% of the membrane‐bound IgM into the supernatant; approximately 10% of the IgM pellets with the cytoskeletal fraction on centrifugation. Ligand challenge with either m̈‐chain‐specific antibodies or concanavalin A induces a change in the state of membrane IgM making it refractory to detergent extraction, such that 43% of the IgM pellets during centrifugation. This ligand‐induced retention of IgM is significantly diminished by the microfilament‐disrupting agent cytochalasin D, whereas pretreatment of cells with sodium azide or colchicine results in no significant change in the percentage of membrane IgM retained by Triton X‐100 residues. These results indicate that retention of IgM involves an association with the cortical actin‐based cytoskeleton. Investigation of the structural basis for ligand‐induced Triton X‐100 retention of membrane IgM by using ferritin‐conjugated antibodies, myosin subfragment S1, and stereo‐imaging electron microscopy has revealed linkages between ligand‐receptor (antigen‐IgM) complexes and elements of the cortical actin‐based cytoskeleton.
Accumulation of fluorescently labeled actin in the cortical layer in sea urchin eggs after fertilization
Authors: Hamaguchi Yukishisa , Mabuchi Issei
Published: February 2005

Actin from sea urchin eggs was fluorescently labeled with fluorescein isothiocyanate (FITC), N‐(7‐dimethylamino‐4‐methylcoumarinyl)‐maleimide (DACM), or 5‐iodoacetamidofluorescein (IAF) and microinjected into sea urchin eggs and oocytes. It distributed evenly in the cytoplasm of unfertilized eggs. Upon fertilization, actin accumulated first around the sperm binding site and, soon afterwards, in the fertilization cone. The accumulation propagated all over the cortex after a latent period of 10–20 sec. In the case of Clypeaster japonicus eggs, propagation of the accumulation coincided with a shape change in the egg, suggesting that the accumulated actin in the cortex generates forces. FITC‐actin was incorporated into microvilli and retained in the cortex after cleavage. On the other hand, DACM‐ or IAF‐actin was not incorporated into microvilli and was dispersed from the cortex by cleavage. These differences may be attributable to differences in the properties of the actins labeled at different sites. After photobleaching by laser light irradiation, FITC‐ or IAF‐actin redistributed in the cortex of fertilized egg as quickly as it did before fertilization. When an unfertilized egg was injected with both actin and a calcium buffer (intracellular free Ca2+ concentration 9 μM), the actin accumulation was similar to that during fertilization but without the latent period. This suggests that the accumulation depended on the increase in the intracellular free Ca2+ concentration. When the unfertilized egg was injected with 0.2 M EGTA after injection of labeled actin and then inseminated, it accumulated only in the protrusion of cytoplasm where the sperm had entered, and fertilization was not completed. In immature oocytes, the accumulation was observed in the cortical region, including the huge protrusion of the cytoplasm where the sperm had entered. These results suggest that actin accumulation in the sperm binding site plays an important role in the sperm reception mechanism of the egg.
Stepwise sliding disintegration of tetrahymena ciliary axonemes at higher concentrations of ATP
Authors: Tanaka Mikako , Miki‐Noumura Taiko
Published: February 2005

Several characteristics of the sliding disintegration of Tetrahymena ciliary axonemes were found by turbidimetric assay, the ATP‐regenerating system, and quantitative determination of the ATP concentration. At ATP concentrations exceeding 40 μM, the response in terms of turbidity was biphasic and could be divided into three phases. The dependence of each phase on ATP concentration was examined. The time duration of phase 2 increased with ATP concentration. When the ATP concentration was kept constant by the ATP‐regenerating system, consisting of pyruvate kinase and phosphoenol pyruvate, the time duration of phase 2 increased with the concentration of phosphoenol pyruvate. On examining the change in turbidity with decreasing ATP concentration, the transition from phase 2 to phase 3 was found to occur at an ATP concentration of 40 μM.

Dark‐field and electron microscopy indicated the sliding disintegration to be closely correlated with the degree of tubidity. At phase 1, one or two doublets extruded from most of the axonemes, and disintegration failed to progress during phase 2. At the transition point from phase 2 to 3, at about 40 μM, ATP, other doublets were noted to extrude from the axonemes one after the other, causing turbidity to be minimal by the end of phase 3.

The ATP concentration dependence of stepwise sliding disintegration suggests that each axoneme may possess the ability to regulate doublet microtubule sliding at lower or higher concentrations of ATP. In response to local differences or gradients of ATP concentration along the axoneme, the axonemes may cause localized sliding of doublets, thus subsequently generating active bending movement.