bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of sea urchin egg cytoplasmic dynein in mitotic apparatus studied by using a monoclonal antibody against sea urchin sperm flagellar 21S dynein
Authors: Hisanaga Shin‐Ichi , Tanaka Takeshi , Masaki Tomoh , Sakai Hikoichi , Mabuchi Issei , Hiramoto Yukio
Published: February 2005

A monoclonal antibody against sea urchin (Hemicentrotus pulcherrimus) sperm flagellar 21S dynein was characterized and sued to identify and localized cytoplasmic dynein of sea urchin eggs by the methods of immunoblotting and indirect immunofluorescence microscopy. D57, the monoclonal antibody used in this study, was directed to the Aβ polypeptide of 21S dynein. D57 stained sperm flagella specifically but did not inhibit Mg‐ATPase activity of 21S dynein, its recombination ability with NaCl‐extracted axonemes, or the movement of demembranated sperm. D57 cross‐reacted with sea urchin egg cytoplasmic dynein. High molecular weight cytoplasmic dynein polypeptide which had the same electrophoretic mobility s flagellar dynein. A chains was the only polypeptide that reacted with D57 in the crude extract from unfertilized sea urchin eggs. Indirect immunofluorescence observations showed that the mitotic apparatus was stained most intensely in the frozen sections and lysed eggs. In the mitotic apparatus isolated at metaphase, the half spindles were stained more strongly than the astral regions. The regions near chromosomes in the half spindle appeared to be stained particularly. Staining of the interzone was also observed in the mitotic spindle isolated at anaphase. Comparison of the staining patterns for cytoplasmic dynein with that for tubulin suggested that cytoplasmic dynein was localized where microtubules were densely organized, but its distribution may not necessarily be identical with that of microtubules.
Pre‐prophase band of microtubules, absent from tip‐growing moss filaments, arises in leafy shoots during transition to intercalary growth
Authors: Doonan John H. , Cove David J. , Corke Fiona M. K. , Lloyd Clive W.
Published: February 2005

In the presence of cytokinin, undetermined side branch initials of the moss, Physcomitrella patens, arc induced to form buds and then leafy shoots rather than to develop as tip‐growing filaments. This represents a transition between the two modes of plant cell expansion–tip growth and uniform intercalary growth. The organization of microtubules in filaments is different from that in leafy shoots and can be traced back to the influence of phytohormones on side branch initials. Microtubules either focus at a particular region (as in tip‐growing cells) or in the presence of high levels of cytokinin form swollen bud initials in which microtubules are more diffusely organized. Higher levels of cytokinin are capable of destabilizing tip microtubules in caulonemal filaments. Although caulonemata are not normally target cells, this implies that cytokinin may exert its morphogenetic effects by altering microtubule organization.

In tip‐growing filaments, interphase microtubules trace a meandering course through the cytoplasm towards the tip and are not for the main part associated with the plasma membrane as are cortical arrays. There is no pre‐prophase band of microtubules to indicate the future division plane, even though the oblique division plane is known to be precisely controlled relative to environmental factors. This microtubule cycle contrasts with cells of the leafy shoots that develop from buds: in these, the interphase array is cortical, consisting of flat‐pitched microtubular helices that do not focus upon a growing tip. It is now shown that pre‐prophase hands occur at this stage.

The absence of bands does not readily correlate with imprecise control of the division plane. Instead, it is proposed that the ability to form pre‐prophase bands depends upon the arrangement of microtubules in the preceding interphase array. Ways in which bands might be formed are discussed and the generality of these ideas is tested by observations on higher plant cells.
New evidence for a “biased baseline” mechanism for calcium‐regulated asymmetry of flagellar bending
Authors: Eshel Dan , Brokaw Charles J.
Published: February 2005

Time‐averaged data covering six to ten beat cycles for ATP‐reactivated spermatozoa of a sea urchin and Ciona, and from a uniflagellate mutant of Chlamydomonas, were analyzed to obtain parameters of oscillation and mean shear angle at each point along the flagellum. The mean shear angles usually show a sharp change near the base of Ihc flagellum. This sharp basal change in angle is correlated with perceived asymmetry in the development times of principal and reverse bends when these bends are measured directly from the asymmetric bending patterns, without subtracting out the mean shear angle. The asymmetry in development times was previously considered to be evidence against a “biased baseline” mechanism for asymmetric bending waves, in which completely symmetric bending waves develop and propagate on a curved flagellum. Our analysis now shows that the asymmetry in development times can be fully explained by the presence of a sharp static bend near the base of the flagellum, which can confuse the determination of the times of initiation of new bends at the base of the flagellum. Our reinterpretation of these data removes previous objections to the “biased baseline” mechanism for the regulation of bending wave asymmetry by calcium, and supports other evidence favoring a biased baseline mechanism, rather than a “biased switching” mechanism.
Myosin filaments in cytoskeletons of Dictyostelium amoebae
Authors: Clarke Margaret , Baron Andre
Published: February 2005

Cytoskeletons were prepared from vegetative amoebae of Dictyostelium discoideum by extraction with Triton X‐100. The cytoskeletons were suspended in buffers known to induce the assembly or disassembly of myosin filaments. The samples were fixed, and thin sections were examined by transmission electron microscopy. In both types of buffers, myosin‐containing cytoskeletons exhibited a ring of densely staining proteinaceous material within the cortical filament matrix; this ring was not observed in myosin‐free cytoskeletons. When myosin‐containing cytoskeletons were placed in buffers that induced myosin polymerization, the ring appeared as an array of rodlike filaments approximately 13 nm wide and up to 0.5 μm in length—dimensions appropriate for myosin thick filaments. If ATP was added to cytoskeletons containing such filaments, the cytoskeletons contracted and the ring of filaments disappeared. ATP‐induced contraction of cytoskeletons was also visualized by indirect immunofluorescence by using monoclonal antibodies to Dictyosielium myosin. All data were consistent with the identification of the protein ring seen by electron microscopy as cortical myosin. Its location and organization were appropriate for the production of cortical contraction through a sliding filament mechanism.
Protein phosphorylation and dynamics of cytoskeletal structures associated with basal bodies in Paramecium
Authors: Keryer Guy , Davis Frances M. , Rao Potu N. , Beisson Janine
Published: February 2005

The presence of phosphorylated proteins associated with microtubule organizing centers in tissue culture cells during mitosis has been demonstrated by the use of monoclonal antibodies raised against mitotic HeLa cells [Vandre et al., Proc. Natl. Acad. Sci. U.S.A. 81:4439–4443, 1984]. We report here that in Paramecium two of the mitosis specific antibodies, MPM‐1 and MPM‐2, decorate throughtout the cell cycle all the microtubule organizing centers (MTOCs) located in the cortex and in the oral apparatus (gullet). Immuno‐electron microscopy showed that these antibodies labeled the electron‐dense material surrounding basal bodies from which several microtubule networks as well as kinetodesmal fibers originate. During mitosis, these antibodies also stained other cortical cytoskeletal structures, the kinetodesmal fibers (MPM‐1 and MPM‐2) and the epiplasm (MPM‐1). Among the different polypeptides recognized by the antibodies on immunoblots, three major ones of 60, 63, and 116 kDa were found to be common to the cortex (where several thousand ciliary basal bodies are anchored) and the oral apparatus (which comprises several hundred basal bodies around which various arrays of cytoplasmic microtubules are organized). Alkaline phosphatase treatment abolished the immunoreactivity of the polypeptides and the labeling observed by immunofluorescence. These results demonstrate that phosphorylated proteins are associated with all the known active microtubule organizing centers present in the cortex throughout the cell cycle of Paramecium. Furthermore they indicate that in Paramecium phosphorylation of proteins could also be involved in the cell cycle dependent dynamics of cortical cytoskeletal structures other than microtubules.
Bending patterns of Chlamydomonas flagella: IV. Mutants with defects in inner and outer dynein arms indicate differences in dynein arm function
Authors: Brokaw C. J. , Kamiya R.
Published: February 2005

Mutants with outer dynein arm defects or deficiencies all show a major reduction in beat frequency to about half the normal value; some of these mutants show an additional decrease in sliding velocity associated with reduced shear amplitude and an additional reduction in beat frequency, as well as other more minor modifications of the normal forward mode bending pattern. New mutants (ida98, pf30), which appear to be deficient in a subset of inner dynein arms show a reduction in sliding velocity that is primarily associated with a reduction in shear amplitude, with only a small reduction in beat frequency. These differences in motility phenotype between inner and outer dynein arm mutants suggest that inner and outer dynein arms may have distinct functions. The relatively large decrease in sliding velocity associated with partial loss of inner arms is consistent with earlier observations on pf23, a nonmotile mutant lacking inner arms, suggesting that inner arms may have an essential function in motility. The ability to generate reverse mode bending patterns is retained in some inner or outer dynein arm mutants, but appears to be decreased in those mutants which show reduced shear amplitude for the forward mode bending pattern.
Axonal tubulin and microtubules: Morphologic evidence for stable regions on axonal microtubules
Authors: Sahenk Zarife , Brady Scott T.
Published: February 2005

Biochemical studies indicate that axonal tubulin is composed of at least two distinct pools that differ in cold solubility and biochmical composition [Brady et al: J. Cell Biol. 99:1716–1724]. To determine the morphologic correlate of cold‐insoluble tubulin, segemnts of rat optic nerves were exposed to a series of in vitro experimental conditions that affect microtubules (MTs), including cold, podophyllotoxin (PT), triflupromazine (TFP), and taxol, and then examined by electron microscopy. Longitudinal sections of control axons showed MTs oriented parallel to the long axis of the axons. Axond exposed to Cold, PT, and TFP showed short segments of MTs in association with cytoskeletal disarray. Morphometric studies were used to distinguish between a simple malorientation of MTs (undulation or zigzags in their course) and the loss of labile segments of MTs, leaving the stable portions behind. The lengths of MT segments were measured in longitudinal sections, and the numbers of MTs were determined in the cross sections. All MT segment‐length histograms showed a unimodal distribution. Cold and PT produced a simple shift of the control histogram to the shorter length MTs. In cross sections the numbers of MTs in cold‐ and PT‐exposed axons were significantly decreased, indicating that the presence of short segments of MTs. Taxol, an agent that promotes MT assembly, reversed the cold effect partially and resulted in increases in both MT segment lenght and number. These studies indicate that stable MT segments are portions of longer MTs containing both stable and labile regions. Furthermore, these findings are consistent with the hypothesis that cold‐insoluble tubulin functions as a transportable MT‐organizing complex in the axon.
Binding of mammalian brain microtubule‐associated proteins (MAPs) to insect ovarian microtubules
Authors: Stebbings Howard , Anastasi Angela , Indi Shantinath , Hunt Cherryl
Published: February 2005

In this study we have applied microtubule‐associated proteins (MAPs) from mammalian brain to both native and reassembled insect ovarian microtubules. Such microtubules, which are normally smooth walled, become decorated with projections similar to those observed when mammalian brain MAPs are added back to assembling or assembled mammalian brain microtubules. The mammalian MAPs were also detected as components of insect microtubules when analyzed by polyacrylamide gel electrophoresis. Our observations suggest that mammalian brain MAPs have common binding sites on microtubules from two widely different sources and indicate the degree of evolutionary conservation of such sites.
Stimulation of in vitro motility of Chlamydomonas axonemes by inhibition of cAMP‐dependent phosphorylation
Authors: Hasegawa Etsuko , Hayashi Hiroshi , Asakura Sho , Kamiya Ritsu
Published: February 2005

When demembranted axonemes of Chlamydomonas were reactivated with Mg ATP, the proportion of motile axonemes was significantly increased by the preence of either phosphodiesterase (PDE) or protein inhibitor of cAMP‐dependent kinase (PKI). The effect of PDE was cancelled by the addition of cAMP. These findings strongly suggest that the axoneme samples have endogenous cAMP, which can reduce the proportion of motile axonemes via phosphorylation. This inhibitory effect of cAMP on Chlamydomonas axonemes is opposite to its stimulatory effect on the axonemal motility in other organisms so far reported. PKI or PDE activated the motility motility either in the absence of Ca2+, when the axonemes beat with an asymmetric waveform, or in 10−5M Ca2+, when the axonemes beat with a symmetric waveform. This cAMP‐dependent regulation of motility was observed with the axonemes from which detergent‐soluble material had been removed, indicating that the proteins responsible for the regulation still remained in the axonemes. Preliminary in vitro phosphorylation stdies have implicated two polypetides as candidates for the target protein of cAMP‐dependent protein kinase: one with a molecular weight of 270 kD and the other with a much larger molecular weight.
Characterization of villin from the intestinal brush border of the rat, and comparative analysis with avian villin
Authors: Alicea H. A. , Mooseker M. S.
Published: February 2005

The biochemical properties of villin purified from the brush borders of chicken and rat small intestines were compared, with emphasis on their physical properties and their Ca++‐dependent interaction with actin. Like chicken villin, rat villin exists as two isoforms present in equimolar concentrations; the rat isoforms are slightly more acidic than those of chicken villin (6.08 and 6.11 versus 6.26 and 6.34). Rabbit antisera raised against either villin crossreacted with the other one. Like the avian protein, rat villin bundled F‐actin at calcium concentrations below 0.1 μM. Above ∼︁1 μM calcium, it accelerated the rate of actin assembly and restricted filament lengths of F‐actin formed either during coassembly with villin or by addition of villin to preformed filaments. The threshold calcium concentration required for effective severing of preformed filaments was approximately tenfold higher than that required for restricting lengths during coassembly. The extent of filament shortening was proportional to the amount of villin present. At a fixed villin concentration, filament length decreased with increasing [Ca++] over a broad range from 10−7−10−4 M. In general, the mean filament lengths and the dispersion about the mean value were lower in samples where filaments were coassembled with villin than when villin was added to preformed filaments.
Motility of the spirochete leptospira
Authors: Goldstein Stuart F. , Charon Nyles W.
Published: February 2005

Spirochetes are a group of bacteria with a unique ultrastructure and a fascinating swimming behavior. This article reviews the hydrodynamics of spirochete motility, and examines the motility of the spirochete Leptospira in detail. Models of Leptospira motility are discussed, and future experiments are proposed.

The outermost structure of Leptospira is a membrane sheath, and within this sheath are a helically shaped cell cylinder and two periplasmic flagella. One periplasmic flagellum is attached subterminally at either end of the cell cylinder and extends partway down the length of the cell. In swimming cells, each end of the cell may assume either a spiral or a hook shape. Translational cells have the anterior end spiral shaped, and the posterior end hook shaped. In the model of Berg et al., the periplasmic flagella are believed to rotate between the sheath and the cell cylinder. Rotation of the anterior periplasmic flagellum causes the generation of a gyrating spiral‐shaped wave. This wave is believed sufficient to propel the cells forward in a low‐viscosity medium. The cell cylinder concomitantly rolls around the periplasmic flagella in the opposite direction—which allows the cell to literally screw through a gel‐like viscous medium without slippage. This model is presented, and it is contrasted to previous models of Leptospira motility.
51‐kd protein, a component of microtubule‐organizing granules in the mitotic apparatus involved in aster formation in vitro
Authors: Toriyama Masaru , Ohta Kunihiro , Endo Sachiko , Sakai Hikoichi
Published: February 2005

Mitotic apparatuses (MAs) isolated from sea urchin metaphase eggs were chilled on ice to depolymerize microtubules, homogenized, and incubated with tubulin. This caused formation of many small asters with microtubules focusing on granules which were probably fragments of the centrosome. The aster‐forming protein components of the granules in the homogenized MAs were solubilized in 0.5 M KCl containing 50% glycerol. After dialysis against low‐ionic‐strength buffer solution, proteins congregated to form granular assembly capable of initiating aster formation. Phosphocellulose column chromatography enabled the separation of the aster‐forming protein fraction which contained a 51,000 molecular weight protein (51‐kd protein) as a major component. The protein fraction possessing the aster‐forming activity was also prepared from methaphase whole egg homogenate, and the elution profile of the 51‐kd protein on phosphocellulose column also coincided with that of the aster‐forming activity. The granular assembly reconstituted from the phosphocellulose fraction formed asters whose microtubules show the same growth rate and length distribution as those of asters reconstructed from the granules in the homogenized MAs. Anti‐51‐kd protein antibody that was raised in rabbit and affinity‐purified stained the center of asters which were reconstructed either from the granules in the homogenized MAs or from the granular assembly reconstituted from the phosphocellulose fraction. These results suggest that the 51‐kd protein is a component in the aster‐forming activity of the centrosomal component in vitro.
Interaction of chlamydomonas dynein with tubulin
Authors: Haimo Leah T. , Fenton Raymond D.
Published: February 2005

Studies were conducted to determine if dynein could bind to unpolymerized tubulin. Tubulin alone normally fractionated in the included volume of a molecular sieve Bio‐Gel A‐1.5m column. Incubated together, tubulin and dynein coeluted in the void volumn, suggesting that a complex had formed between the two. In addition, immunoelectron microscopy revealed preassembled microtubules were labeled with biotin antibody only when incubated in both dynein and biotinylated tubulin, evidence that dynein with bound biotinylated tubulin had decorated the microtubules. A fraction of the tubulin could be dissociated from dynein by addition of ATP and vanadate, as assayed by molecular sieve chromatography followed by densitometry of gels, suggesting that some tubulin bound to the B end of the dynein arm. Additional tubulin dissociated from the dynein under conditions of high salt. These studies, together with those indicating that tubulin blocked the A end of the dynein arm from binding to microtubules and promoted the interaction of two arms at their A ends, provide evidence that the A end of the arm also can bind tubulin. Thus, the tubulin subunits, themselves, on a microtubule rather than a particular surface lattice structure formed by adjacent protofilaments may provide the binding sites for both ends of the dynein arm.
Subcellular sequestration of an antigenically unique β‐tubulin
Authors: Gallo Jean‐Marc , Précigout Eric , Schrével Joseph
Published: February 2005

Tubulin from Trypanosoma brucei was characterized by Western blotting using well defined monoclonal antibodies reacting with α‐ or β‐tubulin and a new monoclonal antibody, 1B41, raised against a microtubule‐enriched fraction of T. brucei, which specifically reacts with the β‐subunit of tubulin from either T. brucei or rat brain. This antibody has been used to examine the subcellular distribution of the corresponding antigen in T. brucei by indirect immunofluorescence. The epitope recognized by 1B41 is restricted to a thin line extending from the basal body region to the anterior end of the cell body. To determine the relationship between the immunoreactive zone and the flagellum, double‐label immunofluorescence was performed in both interphase and mitotic cells with 1B41 and a flagellar marker, the monoclonal antibody 5E9, specific for the paraflagellar rod polypeptides of trypanosomes. These experiments revealed that the immunoreactive tubulin was contained in a part of the subpellicular cytoskeleton that remained in a constant spatial correspondence with the flagellum throughout the cell division cycle. The β‐tubulin recognized by 1B41 may be segregated into the microtubular structures associated with a cisterna of the endoplasmic reticulum forming the subflagellar microtubule quartet (SFMQ). These results suggest that the presence of an antigenically unique β‐tubulin defines a subpopulation of microtubules possessing specfic dynamic properties that may be involved in the morphogenesis of daughter cells during the division of T. brucei.
Colocalization of F‐actin and 34‐kilodalton actin bundling protein in dictyostelium amoebae and cultured fibroblasts
Authors: Johns Julie A. , Brock Alice M. , Pardee Joel D.
Published: February 2005

The Ca+2‐sensitive actin‐binding protein isolated from Dictyostelium discoideum, 30,000‐D protein (Fechheimer and Taylor: J. Biol. Chem. 259:4514–4520, 1984;) has recently been localized in filipodia of substrate‐adhered amoebae (Fechheimer: J. Cell Biol. 104:1539–1551, 1987). We have determined that this protein has a Mr of 34,000 daltons and is strictly colocalized with actin filaments in both substrate‐attached Dictyostelium amoebae and cultured fibroblasts. 3T3 fibroblasts, as well as normal and virally transformed rat kidney fibroblasts (NRK) contain a 34‐kilodalton (kD) protein that cross‐reacts specifically with antibody to the Dictyostelium bundling protein. Mammalian 34‐kD protein is colocalized with F‐actin in stress fibers and the cortical cytoskeleton in substratadhered fibroblasts. In substrate‐adhered vegetative Dictyostelium, F‐actin and 34‐kD protein are concentrated in regions of the cell cortex exhibiting filipodia and membrane ridges. Multiple filipodia formed after exposure to the chemoattractant folic acid stain intensely for 34‐kD protein, implying participation in the assembly of actin bundles during filipod formation. The cortex of pseudopodia also contained high concentrations of bundling protein, but pseudopod interiors did not. In contrast to vegetative Dictyostelium, F‐actin and 34‐kD protein were not colocalized in cells that had progressed through the development cycle. In fruiting bodies, 34‐kD protein was detected by immunofluorescence microscopy only in prespore cells, while F‐actin appeared in stalk cells and spores.
Axonal neurofilaments differ in composition and morphology from those in the soma of the squid stellate ganglion
Authors: Tytell M. , Zackroff R. V. , Hill W. D.
Published: February 2005

Triton X‐100 insoluble neurofilament (NF) fractions were obtained from two parts of the stellate ganglion and the main giant axon. These were analyzed by one‐ and two‐dimensional gradient polyacrylamide gel electrophoresis, cyclic assembly and disassembly, and electron microscopy. The NF fractions from the ganglion cell bodies (GCB) and from the part of the ganglion mainly consisting of axon initial segments (GIS) were of similar composition; neither contained detectable amounts of the 220 kda and high molecular weight ( 400 kda) NF subunits that were prominent in the axonal NF fraction. However, the GCB and GIS did contain large quantities of a set of 65 kda polypeptides that were minor constituents of the axonal NF fraction. The 65 kda‐containing NF fraction from the ganglion could be cyclically disassembled and reassembled, but only under low salt conditions, in contrast to the high salt conditions used to cycle axonal NFs. A comparison of the peptide map of the 65 kda polypeptides with that of the 60 kda axonal NF subunit showed them to be different. These biochemical differences between the ganglionic and axonal NF fractions correlated with morphologic distinctions: ganglionic NFs were relatively smooth surfaced, whereas axonal NFs had long sidearms. Such observations support the hypothesis that the NF cytoskeleton of the neuron soma is different from that of the axon. Furthermore, the change from the somal form to the axonal form of NFs appears to occur in the region where the axon initial segment increases in diameter to become the axon proper.
Microtubule‐associated organelle and vesicle transport in fibroblasts
Authors: Hayden John H.
Published: February 2005

Allen Video‐enhanced constrast/differential interference constrast (AVEC‐DIC) microscopy was used in conjunction with video intensification immunofluorescence microscopy to demonstrate that organelles and vesicle (particles) can move in either direction along microtubular linear elements in fibroblasts [Hayden et al., 1983]. Since it is not possible to determine the number of microtubules making up a linear element with light microscopy alone, AVEC‐DIC microscopy was used in conjunction with whole‐mount electron microscopy to show bidirectional transport along a single microtubule [Hayden and Allen, 1984]. These studies demonstrate that the structural polarity of the microtubule does not determine the direction of particle motion, and since dynein is an asymetric molecule, a simple microtubule‐dynein‐particle hypothesis cannot explain bidirectional transport along a single microtubule.

Very little is known about regulation of particle transport in most cell types. Human embryonic lung fibroblasts grown on glass coverslips were serum‐deprived for 24 hours and re‐fed with serumless medium; the particle translocations/5 minutes were then determined The cells were then re‐fed with either serumless medium, serum‐containing medium, or serumless medium containing some bioactive factor, and the particle translocations/5 minutes were again determined for the same cells. Medium containing 10% fetal bovine serum inhibited particle translocation by 51.8%. Of the bioactive factors tested, only vasopressin produced a significant reduction in particle translocations (38%). This suggests that protein kinase C or calcium/calmodulin kinase could be involved in regulating particle transport.
Two monoclonal antibodies to actin: One muscle selective and one generally reactive
Authors: Lessard James L.
Published: February 2005

Two IgG1, κ monoclonal antibodies (Mab) against actin have been obtained from a fusion in which chicken gizzard actin was used as the immunogen. One Mab, designated B4, shows a preferential reactivity toward enteric smooth muscle actin but also cross‐reacts with skeletal, cardiac, and aorta actins on the basis of immunoblots, ELISA assays, and indirect immunofluorescence. However, this antibody does not react with either cytoplasmic actin in any of these assay systems. A second Mab, designated C4, reacts with all six known vertebrate isoactins as well as Dictyostelium discoideum and Physarum polycephalum actins. Thus B4 Mab appears to react with an epitope that is at least partially shared among the muscle actins but not found in cytoplasmic actins, while C4 Mab binds to an antigenic determinant that has been highly conserved among the actins. The binding sites of both Mabs on skeletal actin overlap that of pancreatic DNase I. Both antibodies bind a SV8 proteolytic product comprising the amino‐terminal two‐thirds of the actin molecule, and their epitopes appear to overlap since C4 can compete for the binding of B4 to skeletal actin. Neither antibody is able to prevent actin polymerization.
Colocalisation of acetylated microtubules, glial filaments, and mitochondria in astrocytes in vitro
Authors: Cambray‐Deakin Martin A. , Robson Susanne J. , Burgoyne Robert D.
Published: February 2005

We have recently shown that acetylated α‐tubulin containing microtubules (acety1‐MTs; labeled by antibody 6‐11B‐1) constitute a cold‐stable subset of the microtubule network of nonneuronal cells in rat primary forebrain cultures [Cambray‐Deakin and Burgoyne: Cell Motil. 8(3):284–291, 1987b]. In contrast, tyrosinated α‐tubulin containing MTs (tyr‐MTs; labeled by antibody YL1/2) are cold‐labile. Here we have examined the distribution of acety1‐MTs and tyr‐MTs in cultures of newborn rat forebrain astrocytes and simultaneously investigated the distribution of mitochondria and glial filaments. In double‐label immunofluorescence experiments a marked colocalisation of acetyl‐MTs and glial filament bundles was observed. Tyr‐MTs did not show a similar colocalisation with glial filament bundles. Furthermore, the distribution of mitochondria closely followed that of the acetyl‐MT and glial filament bundles. When cells were exposed to short‐term (30‐min) treatments with MT‐disrupting agents such as colchicine and nocodazole, the tyr‐MT network was removed but the distributions of acetyl‐MTs, glial filaments, and mitochondria were unchanged. Increased exposure to colchicine (9–16 hr) caused a progressive disruption of the acetyl‐MTs and the collapse of glial filaments and mitochondria to the perinuclear region. These results suggest that acetyl‐MTs and glial filaments but not tyr‐MTs may be involved in the intracellular transport of organelles and/or in the control of their cytoplasmic distribution.
Iontophoretic localization of Ca‐sensitive sites controlling activation of ciliary beating in macrocilia of Beroë: The ciliary rete
Authors: Tamm Sidney L.
Published: February 2005

Macrocilia are thick compound ciliary organelles found on the lips of the ctenophore Beroë. Each macrocilium contains several hundred axonemes enclosed by a single common membrane around the shaft of the organelle. Macrocilia are activated to beat rapidly and continuously in the normal direction by stimulus‐triggered Ca influx through voltage‐dependent Ca channels (Tamm, 1988). Heat‐dissociated macrociliary cells are spontaneously active without depolarizing stimuli, providing Ca is present (Tamm, 1988). Here we investigate the spatial distribution of macrociliary Ca channels by iontophoretic application of extracellular Ca to different sites along quiescent, “potentially activated” macrocilia of dissociated cells in Ca‐free medium. We find that Ca sensitivity for eliciting motility is highest or resides exclusively on the basal portion of the macrociliary surface. This is the first demonstration of local differences in Ca morphologically with a reticulum of unfused ciliary membranes at the base of the macrocilium. This ciliary rete is in direct communication with the surrounding sea water. It is likely that the ciliary rete provides the necessary Ca influx to trigger beating by virtue of its greater Ca conductance (i.e., density of Ca channels) and/or greater total membrane area.
Video supplement assembly, disassembly, and movements of the microfilament‐rich ridge during the amoeboflagellate transformation in Physarum polycephalum
Authors: Pagh Kathryn I. , Adelman Mark R.
Published: February 2005

The amoeboflagellate transformation in Physarum polycephalum involves a series of dramatic changes in cell shape and motile behavior. This report describes the morphological and behavioral changes through which a synchronously transforming population of cells passes, stressing that, although there are a series of distinguishable stages, cells at all stages display striking plasticity. Our previous studies showed that amoeboflagellates transiently display a flattened motile extension—the ridge—that projects from a specific location on the cell surface and contains a laminar core densely packed with a series of crisscrossing arrays of actin microfilaments. Details are presented here concerning the movements of the ridge as well as the dynamics of ridge formation and disassembly in relation to other morphogenetic events of the transformation. The ridge forms at about the same time as transforming cells begin to elongate, propagates undulations parallel to the long axis of the cell as the transformation proceeds, and disassembles late in the transformation. Staining of fixed cells with the fluorescent probe rhodaminephalloidin shows that the actin of amoeboid cells is strikingly redistributed as the transformation proceeds. Amoeboflagellates contain most of the stainable actin in the ridge and in a ventral‐posterior spot that may be a site of cell‐substratum adhesion. These results provide additional insights into the possible functions of the ridge and the roles of actin during the amoeboflagellate transformation.
Isolation of an immunoreactive analogue of brain fodrin that is associated with the cell cortex of Dictyostelium amoebae
Authors: Bennett Holly , Condeelis John
Published: February 2005

We have used a polyclonal affinity‐purified antibody made against chicken brain fodrin (both 240 and 235 Kd subunits) as a probe to determine if a fodrinlike protein exists in amoebae of Dictyostelium discoideum. In Western blots of whole cells and the isolated cell cortex, polypeptides measuring 220 and 70 Kd are recognized by the fodrin antibodies. In situ localization by indirect immunofluorescence with antifodrin indicates that the immunoreactive polypeptides are cortical. The immunoreactive analogues copatch and cocap with concanavalin A. At the level of resolution of the electron microscope, immunocytochemistry with antifodrin and colloidal gold confirms that the immunoreactive analogues are cortical proteins associated with microfilaments on the cytoplasmic side of the plasma membrane. We have isolated and characterized the 220 Kd protein to determine if it is similar to fodrin and to investigate its relationship to the 70 Kd polypeptide. The 220 Kd protein can be extracted from the cortex in the absence of detergent and isolated by gel filtration and sucrose density gradient sedimentation. The 220 Kd is a rod‐shaped protein 118 ± 17.8 nm (N = 37) in length. It has a sedimentation coefficient of 9.3 S and Stokes' radius of 13 nm and exists as a dimer of approximately 500,000 daltons (Mr). Isolated 220 Kd binds to actin filaments in vitro when assayed by rotary shadowing. Morphological criteria distinguish 220 Kd from Dictyostelium myosin II heavy chain (215 Kd) and the filaminlike protein at 240 Kd. The 70 Kd polypeptide appears to be a cleavage fragment of the 220 Kd, since it is found after prolonged storage when formerly only the 220 Kd was present. Furthermore, the 220 and 70 Kd polypeptides exhibit similar one‐dimensional peptide maps when treated with TPCK trypsin. On the basis of its physical and immunoreactive characteristics, and location in the cell, the 220 Kd may be a fodrinlike protein.
Binding of kinesin to stress fibers in fibroblasts under condition of microtubule depolymerization
Authors: Okuhara Koji , Murofushi Hiromu , Sakai Hikoichi
Published: February 2005

The localization of kinesin in EBTr (bovine embryonic trachea fibroblast) cells was studied by indirect immunofluorescence microscopy using an affinity‐purified antibody against bovine adrenal kinesin.

It has already been shown that in interphase cells a part of kinesin is located on microtubules and the rest diffusely distributed throughout the cytoplasm [Murofushi et al., 1988]. When microtubules were depolymerized with cold or colchicine treatment, antikinesin antibody‐stained fibrous components distinct from microtubules. These fibrous structures were considered to be stress fibers because they were stained with rhodamine‐phalloidin and because the fibrous staining with antikinesin antibody was completely lost by treating the cells with cytochalasin D along with colchicine. When cold‐treated cells in which a major part of kinesin had been localized on stress fibers were incubated at 37°C, kinesin reappeared on reconstituted microtubules. These observations strongly suggest that kinesin has affinity not only to microtubules but also to stress fibers in culture cells.
Actomyosin organization during cytokinesis: Reversible translocation and differential redistribution in Dictyostelium
Authors: Kitanishi‐Yumura Toshiko , Fukui Yoshio
Published: February 2005

Synchronized cultures of Dictyostelium discoideum were used to study organizational changes of the cytoskeleton during mitotic cell division. The agar‐overlay technique (Yumura et al.: J. Cell Biol. 99:894–899, 1984) was employed for immunofluorescence localization and video microscopic observation of living mitotic cells. The mitotic phase was defined by changes in chromosome configuration by using a double stain with the fluorescent dye DAPI.

This study showed that the actin‐ and myosin‐containing cytoskeleton was reversibly redistributed between the cortical ectoplasm and the endoplasm during prophase and telophase. Both actin and myosin filaments were dissociated from the cell cortex in prophase. Most of the actin and myosin was filamentous and remained in the endoplasm until telophase. Saltatory movements of organelles stopped suddenly, coincident with the breakdown of the cytoplasmic microtubule network. This change in the microtubule system was temporally coupled with the disappearance of actomyosin from the cortex. At the same time, the local vibrating movement of particles almost stopped, suggesting that the viscoelastic nature of the endoplasm was altered. In the late anaphase, actin and myosin relocalized to the cortical ectoplasm. Early in this phase, myosin filaments were localized specifically at the anticipated cleavage furrow region of the cleavage furrow, whereas actin filaments were redistributed more uniformly in the cell cortex, with an extremely large accumulation in the polar pseudopods. Subsequently the actin formed an orderly parallel array of cables along with myosin filaments in the contractile ring.

The spatial segregation of actin and myosin in late anaphase was clearly demonstrated by multipolar cell division of artificially induced giant cells. Actin was relocalized in both the polar and the proximal constricting regions whereas myosin was only localized in the center of each pair of daughter microtubule networks where the cleavage furrow was formed. This study demonstrates that actin and myosin are reorganized by a temporally coordinated but spatially different mechanism during cytokinesis of Dictyostelium.
Direct visualization of bipolar myosin filaments in stress fibers of cultured fibroblasts
Authors: Svitkina Tatjana M. , Surguchova Irina G. , Verkhovsky Alexander B. , Gelfand Vladimir I. , Moeremans Marc , De Mey Jan
Published: February 2005

The authors examined the molecular organization of myosin in stress fibers (microfilament bundles) of cultured mouse embryo fibroblasts. To visualize the organization of myosin filaments in these cells, fibroblast cytoskeletons were treated with gelsolin‐like protein from bovine brain (hereafter called brain gelsolin), which selectively disrupts actin filaments. As shown earlier [Verkhovsky et al., 1987], this treatment did not remove myosin from the stress fibers. The actin‐free cytoskeletons then were lightly sonicated to loosen the packing of the remaining stress fiber components and fixed with glutaraldehyde.

Electron microscopy of platinum replicas of these preparations revealed dumbbell‐shaped structures of approximately 0.28 μm in length, which were identified as bipolar myosin filaments by using antibodies to fragments of myosin molecule (subfragment I and light meromyosin) and colloidal gold label. Bipolar filaments of myosin in actin‐free cytoskeletons were often organized in chains and lattices formed by end‐to‐end contacts of individual filaments at their head‐containing regions. Therefore, after extraction of actin, it was possible for the first time to display bipolar myosin filaments in the stress fibers of cultured cells.
Myofibril assembly is linked with vinculin, α‐actinin, and cell‐substrate contacts in embryonic cardiac myocytes in vitro
Authors: Terai Masaru , Komiyama Masatoshi , Shimada Yutaka
Published: February 2005

The relationship of nascent myofibrils with the accumulation of adhesion plaque proteins and the formation of focal cell contacts was studied in embryonic chick cardiac myocytes in vitro. The cultures were double‐stained with various combinations of the specific antiactin drug phalloidin and antibodies against vinculin, α‐actinin, connectin (titin), myosin heavy chain, fibronectin, and desmin and examined under fluorescence and interference reflection microscopy.

In the areas of myofibril assembly, vinculin and α‐actinin plaques were formed at the ventral sarcolemmae. These areas overlapped with the sites of cell‐to‐substrate focal contacts and extracellular fibronectin. Because the myofibrils always ran in a straight line between these sites, polarized lines appeared to be generated within the cells in response to their physical (e.g., stress) and/or biochemical environment (e.g., adhesion plaque proteins). The possible presence of other factors cannot be ruled out for the proper alignment of myofibrils. As soon as myofibrils came to span between these adhesion sites, they exhibited typically mature cross‐striated characteristics. Thus, the formation of these inferred lines has some relation to or is in fact necessary for the maturation of myofibrils, in addition to the directional arrangement of sarcomeric proteins.

Additionally, synthesis and distribution of myosin and connectin were tightly linked during early developmental (premyofibril and myofibril) stages. The spatial deployment of desmin was not coupled with vinculin. Thus, connectin and desmin do not appear to form the initial scaffold of sarcomeres.
Use of a novel Chlamydomonas mutant to demonstrate that flagellar glycoprotein movements are necessary for the expression of gliding motility
Authors: Bloodgood Robert A. , Salomonsky Nancy L.
Published: February 2005

As an alternative to swimming through liquid medium by the coordinated bending activity of its two flagella, Chlamydomonas can exhibit whole cell gliding motility through the interaction of its flagellar surfaces with a solid substrate. The force transduction occurring at the flagellar surface can be visualized as the saltatory movements of polystyrene microspheres. Collectively, gliding motility and polystyrene microsphere movements are referred to as flagellar surface motility. The principal concanavalin A binding, surface‐exposed glycoproteins of the Chlamydomonas reinhardtii flagellar surface are a pair of glycoproteins migrating with apparent molecular weight of 350 kDa. It has been hypothesized that these glycoproteins move within the plane of the flagellar membrane during the expression of flagellar surface motility. A novel mutant cell line of Chlamydomonas (designated L‐23) that exhibits increased binding of concanavalin A to the flagellar surface has been utilized in order to restrict the mobility of the concanavalin A‐binding flagellar glycoproteins. Under all conditions where the lateral mobility of the flagellar concanavalin A binding glycoproteins is restricted, the cells are unable to express whole cell gliding motility or polystyrene microsphere movements. Conversely, whenever cells can redistribute their concanavalin A binding glycoproteins in the plane of the flagellar membrane, they express flagellar surface motility. Since the 350 kDa glycoproteins are the major surface‐exposed flagellar proteins, it is likely that most of the signal being followed using fluorescein isothiocyanate (FITC)‐concanavalin A is attributable to these high molecular weight glycoproteins. Therefore, it is likely that the 350 kDa glycoproteins are the ones that must move laterally in the plane of the flagellar membrane in order for the cell to express whole cell gliding motility and microsphere movements along the flagellar surface. This study represents one of the first demonstrations, in any cell type, that whole cell locomotion requires glycoprotein movement within the plane of the plasma membrane.
Changes in the association of actin‐binding proteins with the actin cytoskeleton during chemotactic stimulation of Dictyostelium discoideum
Authors: Dharmawardhane Suranganie , Warren Vivien , Hall Anne L. , Condeelis John
Published: February 2005

Triton‐insoluble cytoskeletons were isolated from Dictyostelium discoideum AX3 cells prior to and following stimulation with 2′deoxy cyclic adenosine monophos‐phate (cAMP). Temporal changes in the content of actin and a 120,000 dalton actin‐binding protein (ABP‐120) in cytoskeletons following stimulation were monitored. Both actin and ABP‐120 were incorporated into the cytoskeleton at 30–40 seconds following stimulation, which is cotemporal with the onset of pseudopod extension during stimulation of amoebae with chemoattraciants. Changes in the content of total cytoskeletal protein and cytoskeletal myosin were determined under the same experimental conditions as controls. These proteins exhibited different kinetics from those of cytoskeletal ABP‐120 and actin following the addition of 2′deoxy cAMP. The authors concluded that the association of ABP‐120 with the cytoskeleton is regulated during cAMP signalling. Furthermore, these results indicate that ABP‐120 is involved in cross‐linking newly assembled actin filaments into the cytoskeleton during chemoattractant‐stimulated pseudopod extension.
Immunocytochemical studies with antibodies to three proteins prominent in the isolated microtubule cytoskeleton of a trypanosomatid
Authors: Bramblett Gregory T. , Kambadur Ravi , Flavin Martin
Published: February 2005

The cytoskeleton of Crithidia fasciculata consists of a corset of paralle microtubules enclosing the cell body and closely underlying the plasma membrane. Distinct sets of crosslinks appear to connect tubules to each other and to membrane. Our objective is to determine the composition of these crosslinks and to elucidate the basis of this spectacular example of membrane‐microtubule interaction. We purified three proteins (designated COP‐33, ‐41, and ‐61 by their subunit Mr), which were consistently abundant in highly purified cytoskeletons. All three bound strongly to microtubules in vitro, and the first two induced bundles through periodic crosslinking. Polyclonal antibodies against each have been used to try to localize these proteins in thin sections of cells or whole mounts of cytoskeletons. Antibodies to COP‐41 bound sepcifically to glycosomes, organelles that encapsulate many glycolytic enzymes in these protozoa, and COP‐41 has been identified as glyceraldehyde 3‐P dehydrogenase.