bluuebunny/crossref_metadata_2025 huggingface dataset:
Microtubules in ascidian eggs during meiosis, fertilization, and mitosis
Authors: Sawada Tomo‐o , Schatten Gerald
Published: February 2005

The sequential changes in the distribution of microtubules during germinal vesicle breakdown (GVBD), fertilization, and mitosis were investigated with antitubulin indirect immunofluorescence microscopy in several species of ascidian eggs (Molgula occidentalis, Ciona savignyi, and Halocynthia roretzi). These alterations in microtubule patterns were also correlated with observed cytoplasmic movements. A cytoplasmic latticework of microtubules was observed throughout meiosis. The unfertilized egg of M. occidentalis had a small meiotic spindle with wide poles; the poles became focused after egg activation. The other two species had more typical meiotic spindles before fertilization. At fertilization, a sperm aster first appeared near the cortex close to the vegetal pole. It enlarged into an unusual asymmetric aster associated with the egg cortex. The sperm aster rapidly grew after the formation of the second polar body, and it was displaced as far as the equatorial region, corresponding to the site of the myoplasmic rescent, the posterior half of the egg. The female pronucleus migrated to the male pronucleus at the center of the sperm aster. The microtubule latticework and the sperm aster disappeared towards the end of first interphase with only a small bipolar structure remaining until first mitosis. At mitosis the asters enlarged tremendously, while the mitotic spindle remained remarkably small. The two daughter nuclei remained near the site of cleavage even after division was complete. These results document the changes in microtubule patterns during maturation in Ascidian oocytes, demonstrate that the sperm contributes the active centrosome at fertilization, and reveal the presence of a mitotic apparatus at first division which has an unusually small spindle and huge asters.
Distribution of acetylated α‐tubulin in retina and in in vitro—assembled microtubules
Authors: Sale Winfield S. , Besharse Joseph C. , Piperno Gianni
Published: February 2005

We have used the mouse monoclonal antibody 6‐11B‐1, specific for acetylated α‐tubulin, to determine the distribution of acetylated α‐tubulin in in vitro–assembled microtubules and retinal tissue. Analysis by immunoblots revealed that microtubules assembled from bovine brain extracts contain both acetylated and nonacetylated α‐tubulin. Immunofluorescence, using 6‐11B‐1 and antitubulin B‐5‐1‐2, a monoclonal antibody specific for α‐tubulin, demonstrated the colocalization of both α‐tubulin species in neurons of the retina and that acetylated microtubules are relatively abundant in neurons. However, analysis at higher resolution revealed that rod photoreceptors contain spatially distinct microtubule arrays which differ in content of acetylated α‐tubulin and differ in stability. Acetylated microtubules which composed those of the rod outer segment and connecting cilium were resistant to depolymerization in nocodazole or colchicine. In contrast, the nonacetylated microtubules which composed those of the rod‐inner segment were depolymerized in nocodazole or colchicine. Therefore, these acetylated microtubules are more resistant to depolymerization than non‐acetylated microtubules.
Microtubule rearrangements during mitosis in multinucleate cells
Authors: Armas‐Portela R. , Paweletz N. , Zimmermann H.‐P. , Ghosh S.
Published: February 2005

The peroxidase‐antiperoxidase (PAP) method for the detection of polymerized tubulin has been used to study the microtubule rearrangements during mitosis in PtK1 and HeLa multinucleate cells obtained by polyethyleneglycol (PEG)‐mediated fusion. We demonstrate here that the transition of the microtubular cytoskeleton from interphase to mitosis is an inducible event and independent of the factor(s) responsible for chromatin condensation and nuclear envelope breakdown. However, for the induction of the microtubule rearrangements nuclear envelope breakdown is required. At midprophase, cytoskeletal microtubule rearrangements start for multinucleate PtK1 cells, whereas in HeLa cells such changes are delayed, and a more abrupt transition is observed here. After complete nuclear envelope breakdown (prometaphase) mitotic asters and spindles but no cytoplasmic (interphase) microtubuli can be observed in both systems. Metaphase is characterized by an interaction between the different mitotic poles which show the form of bipolar spindles, but individual separated mitotic poles far removed from the chromatin can also be seen.
Comparative study of the beat patterns of american and asian horseshoe crab sperm: Evidence for a role of the central pair complex in forming planar waveforms in flagella
Authors: Ishijima Sumio , Sekiguchi Koichi , Hiramoto Yukio
Published: February 2005

American horseshoe crab sperm flagella have the typical 9+2 structure whereas Asian horseshoe crab sperm flagella have a 9+0 axoneme lacking central pair and central sheaths. Beat patterns of the American and the Asian horseshoe crab sperm were recorded by means of a high‐speed video system (200 fields/second) and were compared in order to study the role of the central pair of the axoneme in ciliary and flagellar movement.

The American horseshoe crab sperm beat with relatively planar waves, whereas the Asian horseshoe crab sperm beat with right‐handed helical waves. These results suggest that the central complex plays an important role in forming planar waves, whereas it is not essential for the conversion of microtubule sliding into axonemal bends.
Cell model contraction in the ciliate spirostomum
Authors: Ishida Hideki , Shigenaka Yoshinobu
Published: February 2005

As the species name indicates, the large heterotrichous ciliate Spirostomum ambiguum is characterized by a twisting contraction of the cell body that is easily triggered by various kinds of external stimuli. On the basis of morphological studies, contraction and extension of this organism have been considered to result from antagonistic actions of myoneme and microtubular ribbons. After many trials, we have succeeded in preparing cell models to examine induced contractions and extensions of the cell body. The contraction of this model was induced by increasing the free Ca2+ concentrations even in the absence of Mg‐ATP and was reversed by adding Mg‐ATP without Ca2+. Using dynein ATPase inhibitors such as vanadate and ATP analogs, furthermore, the experiments revealed that the ATPase that generated the force between the two neighboring microtubular ribbons might be a dynein‐like ATPase.
Cytochalasin‐Induced reorganization of actin in allium root cells
Authors: Palevitz Barry A.
Published: February 2005

The effect of cytochalasins on F‐actin was investigated in Allium root cells stained with rhodamine‐phalloidin. With cytochalasin D (CD), the normal interphase network of actin fibers is replaced by dispersed rods and specks similar to those seen in animal cells. However, during division, the specks accumulate at the poles in the form of one to a few large aggregates. The effects intensify with increasing concentration (0.5–5 m̈g/ml) and exposure time (0.5–3 hr). Further, similar behavior is observed with cytochalasin B, but dihydroCB has little effect. Double localizations show that during preprophase, aggregates cluster in association with microtubule foci at the new poles located near the nuclear envelope. From metaphase through anaphase, the aggregates are often found near the tips of kinetochore fibers, while in telophase they are often appressed to the pole side of the daughter nuclei. No association is seen between actin and the other microtubule arrays. The reorganization of F‐actin into small specks is unaffected by sodium azide, but aggregation at the poles is very sensitive to this agent. Polar clustering is also blocked by oryzalin, colchicine, and isopropyl n‐(3‐chlorophenyl) carbamate, but taxol has no effect. Experiments with scleroderma serum 5051 show that CD‐induced aggregates are embedded in centrosomal material at the poles. The results reveal that the reorganization of actin in response to cytochalasins differs during the cell cycle. Furthermore, the aggregation of actin during division is probably governed by an energy‐dependent interaction with microtubules.
Developmental organization of the intestinal brush‐border cytoskeleton
Authors: Takemura Reiko , Masaki Tomoh , Hirokawa Nobutaka
Published: February 2005

At the terminal web of the chicken intestinal epithelial cell, the actin bundles are cross‐linked by a fine filamentous network of actin‐associated cross‐linkers. Myosin, fodrin, and TW 260/240 have been identified as major components of the cross‐linkers. We studied the development of the cross‐linkers by quick‐freeze, deep‐etch electron microscopy, and the expression of cross‐linker proteins (myosin, fodrin 240, and TW 260) by immunofluorescence and immunoblotting analysis during the embryogenesis. Microvilli start to form at 5–7 days, and the rootlets begin to elongate at 10 days. At an early stage of the development of the terminal web (13 days), fodrin 240 and a small amount of myosin are expressed, and a few actin‐associated cross‐linkers are present between the rootlets. However, TW 260 is not expressed at this stage. At an intermediate stage (19 days), the amount of myosin increases, and TW 260 begins to be expressed. The number of cross‐linkers associated with the unit length of the rootlets is 24/μm. At the final stage of the terminal web formation (2 days after hatching), the amount of fodrin 240, myosin, and TW 260 is similar to the adult level, and the number of the actin‐associated cross‐linkers per unit length of the rootlet is 27/μm (∼︁85% of the adult). These results suggest that the synthesis of cross‐linker proteins may be intricately regulated to achieve the desired density of cross‐linkages at each developmental stage: at early and intermediate stages, sufficient and not an excess of cross‐linkages are formed; and at a final stage, a higher complexity of cross‐linkages is achieved. In addition, there is a differential expression of the components of the actin‐associated cross‐linkers: myosin and fodrin could be early components of the cross‐linkers involved in the basic stabilization of the terminal web structure, whereas TW 260/240 becomes incorporated later, possibly involved in the stabilization preparatory to the rapid elongation of microvilli, which occurs after the formation of the terminal web.
Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle
Authors: Otey C. A. , Kalnoski M. H. , Bulinski J. C.
Published: February 2005

In vertebrate skeletal muscle, the proliferating myoblasts synthesize nonmuscle isoforms of actin, and the cells begin to express muscle‐specific actin isoforms during their myogenic differentiation. To study the distributions of the actin isoforms in myogenic cells and fully differentiated skeletal muscle, we prepared a peptide antibody specific for the skeletal α isoform of actin and used this antibody along with an antibody specifically reactive with nonmuscle γ actin to stain cultured myotubes and adult skeletal myofibrils by double‐indirect immunofluorescence. At this level of resolution, no differences in isoform localization were seen: Both muscle and nonmuscle actins were detected in the myotubes and in the striations of mature myofibrils. Myotubes were also double‐stained using immunogold electron microscopy, and the isoform distributions were determined quantitatively by counting the two sizes of gold particles that corresponded to labeling with each antibody. A quantitative analysis of immunoreactivity revealed that, although both forms were present in all actin‐containing structures, nonmuscle actin was relatively more prevalent along the edges (cortical microfilaments) of the myotubes, whereas the muscle isoform predominated in the interior regions (containing forming myofibrils). Thus, we have found evidence of a heterogeneous distribution of muscle and nonmuscle actin isoforms in differentiating myogenic cells, and we have demonstrated that a nonmuscle actin isoform is a component of the muscle contractile apparatus.
Three‐dimensional light microscopy of diploid Drosophila chromosomes
Authors: Agard David A. , Hiraoka Yasushi , Sedat John W.
Published: February 2005

Fluorescence microscopy, uniquely, provides the ability to examine specific components within intact, even living, cells. Unfortunately, high‐resolution conventional fluorescence microscopy is intrinsically a two‐dimensional technique and performs poorly with specimens thicker than about 0.5 μm. Probing the spatial organization of components within cells has required the development of new methods optimized for three‐dimensional data collection, processing, display, and interpretation. Our interest in understanding the relationship between chromosome structure and function has led us to develop the necessary methodology for exploring cell structures in three dimensions. It is now possible to determine directly the three‐dimensional spatial organization of diploid chromosomes within intact nuclei throughout most of the mitotic the cell cycle.
Subcellular compartmentalization by local differentiation of cytoplasmic structure
Authors: Luby‐Phelps Katherine , Taylor D. Lansing
Published: February 2005

The compartmentalization of eukaryotic cells by internal membranes and the subcellular localization of endogenous macromolecules by specific binding mechanisms are familiar concepts. In this report we present evidence that the cytoplasmic ground substance, which surrounds and contains the membranebound compartments, may also be compartmentalized by local differentiations of its submicroscopic structure that sort subcellular particles on the basis of size. The subcellular distribution of size‐fractionated, fluorescent tracer particles was studied in living cells by ratio imaging and fluorescence recovery after photobleaching (FRAP). Large and small particles showed different distributions within the cytoplasmic volume, suggesting that the large particles were relatively excluded from some domains. While the structural basis for this phenomenon is not yet understood in detail, ratio imaging of large and small particles can be used as an empirical tool to identify cytoplasmic compartments for further study. The cytoplasmic diffusion coefficient (Dcyto) and % mobile fraction of the large particles showed considerable spatial variation over the projected area of the cell, while Dcyto and % mobile fraction of the small particles did not. A model is presented to account for this difference. Based on this model, a method is proposed by which FRAP can be used to detect sol‐gel transitions in the cytoplasmic ground substance of living cells.
Results obtained with a sensitive confocal scanning system designed for epifluorescence
Authors: Amos W. B.
Published: February 2005

A wide variety of specimens has been examined with our apparatus, a commercial version of which is being manufactured by Bio‐Rad/Lasersharp. The advantages expected of a confocal system have been realised in practice, the most striking advantage being the exclusion of glare from out‐of‐focus structures. This has made it possible to image cytological details in unflattened cells and intact tissues that were previously inaccessible to the light microscope.
Direct observation of molecular motility by light microscopy
Authors: Harada Yoshie , Yanagida Toshio
Published: February 2005

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

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

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

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

We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23–24°C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half‐time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half‐life of microtubule attachment to the kinetochore is less than several min at 23–24°C.

In the second study, we used high‐resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150‐nm‐thick optical sections that microtubules throughout the spindle laterally associate for several sec into “rods” composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.
Three‐dimensional localization and redistribution of F‐actin in higher plant mitosis and cell plate formation
Authors: Molè‐Bajer Jadwiga , Bajer Andrew S. , Inoué Shinya
Published: February 2005

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

Individual microtubules undergoing treadmilling in vitro were visualized by darkfield light microscopy, and the relationship between treadmilling and dynamic instability was studied as a function of microtubule‐associated proteins (MAPs). In order to demonstrate treadmilling directly by real‐time observation, we constructed three‐block microtubules, the center‐block of which was decorated with Tetrahymena dynein. The decorated block can easily be distinguished from undecorated blocks in the darkfield microscope because the decorated one appears much thicker. At steady‐state conditions, the length of an undecorated block at one end increased and that at another end decreased, while the decorated center‐block did not change in its length. The results from these direct observations show that calf brain 3X‐microtubules exhibit a treadmilling flux of 0.9 μm/h.

Using a similar microscopy technique, we previously demonstrated that phosphocellulose PC‐microtubules existed in either the growing or the shortening phase and alternated quite frequently at steady‐state conditions (dynamic instability). How does treadmilling relate to dynamic instability? An image recording of individual 3X‐microtubules containing MAPs revealed that the microtubules undergo treadmilling and do not exhibit any dynamic instability. This evidence shows that MAPs suppress the dynamic instability of microtubules. That is, treadmilling can take place in the steady state only after microtubules have been stabilized by MAPs.
Detection of single fluorescent microtubules and methods for determining their dynamics in living cells
Authors: Sammak Paul J. , Borisy Gary G.
Published: February 2005

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

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

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

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

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

Free Ca2+ changes the curvature of epididymal rat sperm flagella in demembranated sperm models. The radius of curvature of the flagellar midpiece region was measured and found to be a continuous function of the free Ca2+ concentration. Below 10−7 M free Ca2+, the sperm flagella assumed a pronounced curvature in the same direction as the sperm head. The curvature reversed direction at 2.5 × 10−6 M Ca2+ to assume a tight, hook‐like bend at concentrations of 10−5 to 10−4 M free Ca2+. Sodium vanadate at 2 × 10−6 M blocked flagellar motility, but did not inhibit the Ca2+‐mediated change in curvature. Nickel ion at 0.2 mM and cadmium ion at 1 μM interfered with the transition and induced the low Ca2+ configuration of the flagellum. The forces that maintain the Ca2+‐dependent curvature are locally produced, as dissection of the flagella into segments did not significantly alter the curvature of the excised portions. Irrespective of the induced pattern of curvature, the sperm exhibited coordinated, repetitive flagellar beating in the presence of ATP and cAMP. At 0.3 mM ATP the flagellar waves propagated along the principal piece while the level of free Ca2+ controlled the overall curvature. When Ca2+‐treated sperm models with hooked midpieces were subjected to higher concentrations of ATP (1–5 mM), some cells exhibited a pattern of movement similar to hyperactivated motility in capacitated live sperm. This type of motility involved repetitive reversals of the Ca2+‐induced bend in the midpiece, as well as waves propagated along the principal piece. The free Ca2+ available to the flagellum therefore appeared to modify both the pattern of motility and the flagellar curvature.
Proteolytic fragmentation of Dictyostelium myosin and localization of the in vivo heavy chain phosphorylation site
Authors: Kuczmarski Edward R. , Routsolias Lisa , Parysek Linda M.
Published: February 2005

Dictyostelium myosin was associated into dimers and small oligomers at very low ionic strength, filamentous at intermediate ionic strength, and monomeric in solution conditions of high ionic strength. These different associations were probed by fragmenting myosin with chymotrypsin, trypsin, or V‐8 protease. All three proteases digested monomeric myosin giving rise to multiple fragments with a wide range of molecular weights. Filamentous myosin was not digested by the V‐8 protease, was preferentially cleaved at a single site in the middle of the heavy chain by chymotrypsin, and was cleaved at several sites by trypsin. If the reaction was carried out in very low ionic strength, however, two of these proteases generated stable fragments of high molecular weight. Electron microscopic analysis of these stable fragments showed that tails were shorter than in intact myosin, indicating that the cleavage sites were in the rod portion of the molecule. Under the same conditions of enzymatic digestion, myosin that had been radio labeled in vivo with 32P was analyzed by SDS‐PAGE and autoradiography. By comparing the state of phosphorylation and the size of the stable fragments, it was determined that the heavy chain phosphorylation site was located between 55 and 70 kD from the tip of the myosin tail, near a region where the tail displayed sharp bends.
Characterization of gliding motility inFlexibacter polymorphus
Authors: Ridgway H. F. , Lewin R. A.
Published: February 2005

Motility of the marine gliding bacteriumFlexibacter polymorphuswas studied by using microcinematographic techniques. Following adhesion to a glass surface, multicellular filaments and individual cells usually began to glide within a few seconds at a speed of approximately 12 μm per second (at 23°C). Adhesion to the glass surface was evidently mediated by multitudes of extremely fine extracellular fibrils. Gliding velocity was independent of filament length but directly related to electron‐transport activity and substratum temperature in the range 3–35°C. The rate of gliding was inversely related to medium viscosity, suggesting that the locomotor apparatus functions at constant torque. Forward motion was occasionally interrupted by direction reversals, somersaults (observed primarily in single cells of short filaments), or spinning of filaments tethered by one pole. The frequency of direction reversal was found to be an inverse function of filament length. Translational motility was invariably accompanied by sinistral revolution about the longitudinal axis of a filament. The sense and pitch of revolution were constant among filaments of different length. Polystyrene microspheres or India ink particles adsorbed to gliding cells were actively displaced in either direction, their movement tracing either a regular zigzag or helical path along the filament surface. Because microspheres were also observed to move on nonmotile filaments, particle translocation was evidently not obligatorily linked to gliding locomotion. Multiple particles adsorbed to a single filament often moved independently. The data are consistent with a motility mechanism involving limited motion in numerous mechanically independent (yet functionally coordinated) domains on the cell surface.
The molecular mobility of α‐actinin and actin in a reconstituted model of gelation
Authors: Simon John R. , Furukawa Ruth H. , Ware Bennie R. , Taylor D. Lansing
Published: February 2005

Dictyostelium discoideum α‐actinin (D.d.α‐actinin) is a calcium and pH‐regulated actin‐binding protein that can cross‐link F‐actin into a gel at a submicromolar free calcium concentration and a pH <7 [Fechheimer, et al., 1982]. We examined mixtures of actin and D.d. α‐actinin at four pH and calcium concentrations that exhibited various degrees of gelation or solation. The macroscopic viscosities of these mixtures were measured by falling ball viscometry (FBV) and compared to the translational diffusion coefficients measured by gaussian spot and periodic‐pattern fluorescence photobleaching recovery (FPR) of both the actin filaments and D.d. α‐actinin. A homogeneous, macroscopic gel was not composed of a static actin network. Instead, the filament diffusion coefficient decreased to ∼65% of the control value. If the D.d. α‐actinin concentration was increased, the solution became inhomogeneous, consisting of domains of higher actin concentration. These domains were often composed of a static actin network. The mobility of D.d. α‐actinin consisted of a major fraction that freely diffused and a minor fraction that appeared immobile under the conditions employed. This suggested that D.d. α‐actinin binding to the actin filaments was static over the time course of measurement (∼5 sec). Under solation conditions, there was no apparent interaction of actin with D.d. α‐actinin. These results demonstrate that (1) actin filaments need not be cross‐linked into an immobile, static array in order to have macroscopic properties of a gel, (2) interpretation of the rheological properties of actin:α‐actinin gels are complicated by spatial heterogeneity of the filament concentration and mobility; and (3) a fraction of D.d. α‐actinin binds statically to actin in undisturbed gels. The implications of these results are discussed in relation to cytoplasmic structure and contractility.
Arrangement of desmin intermediate filaments in smooth muscle cells as shown by high‐resolution immunocytochemistry
Authors: Stromer Marvin H. , Bendayan Moïse
Published: February 2005

To gain additional information about the arrangement of intermediate filaments (IF) in normal smooth muscle, fresh avian gizzard was processed for immunoelectron microscopy. The protein A‐gold immunocytochemical technique was applied for the localization of desmin antigenic sites. Desmin‐containing IFs were located in an axial bundle that partially surrounds the nucleus and were associated with numerous mitochondria near the poles of the nucleus. The bundle probably extends the length of the cell. Antibody labeling also showed concentrations of IF around and between cytoplasmic dense bodies (CDB) and also between CDB and membrane‐associated dense bodies (MADB). The relationship between the axial bundle and the nucleus and associated mitochondria suggests that the bundle may support and define the position of these organelles in the cell. A fraying or branching of the bundle may integrate the bundle into the remaining cytoskeletal network of the cell.
Cytokinesis occurs at boundaries of domains delimited by nuclear‐based microtubules in sporocytes of Conocephalum conicum (Bryophyta)
Authors: Brown Roy C. , Lemmon Betty E.
Published: February 2005

Studies of the microtubular cytoskeleton during sporogenesis in the hepatic Conocephalum conicum (Bryophyta) have revealed several unusual phenomena that contribute to understanding the cytokinetic apparatus in plant cell division. Although a typical phragmoplast forms in the interzonal microtubules of the first division spindle and expands to the cell periphery, no cell plate develops. There is no evidence of predetermined division sites and the orientation of both first and second meiotic spindles is imprecise. Simultaneous division of the cytoplasm follows second nuclear division. Equal apportionment of the cytoplasm appears to be a function of the establishment of cytoplasmic domains in the coenocyte, the boundaries of which are delimited by interaction of postmeiotic microtubule systems radiating from the four nuclei. Primary phragmoplasts are initiated in phragmoplasts that are initiated between nonsister nuclei. Depending upon the arrangement of nuclei in the nonpolar sporocyte, from one to three secondary phragmoplasts develop in the zones of contact between opposing sets of microtubules. Except for the site and time of initiation, the two types of phragmoplasts are identical. Eventually the phragmoplasts become confluent and cell plates form in all second division phragmoplasts. It is clear that typical functional phragmoplasts can form in sites determined by interaction of postmeiotic microtubule systems as well as in interzonal spindles as is common in plant cell division.
Effects of taxol on slow and fast axonal transport
Authors: Komiya Yoshiaki , Tashiro Tomoko
Published: February 2005

Axonal transport of tubulin in the rat sciatic nerve is almost completely inhibited by a single subepineural injection of taxol, without affecting that of neurofilament proteins. Actin and a large number of polypeptides cotransported with actin as minor components are also blocked by taxol, although to a lesser extent. Fast axonal transport is essentially free from the inhibitory effect of this drug. Although previous models have suggested that slow axonal transport involves the bulk movement of cytoskeletal structures, these results suggest that such transport may involve an equilibrium between polymerised and depolymerised forms of the axonal cytoskeleton.