bluuebunny/crossref_metadata_2025_split huggingface dataset:
Developmental changes in the arrangement of cortical microtubules in stomatal cells of oat (Avena sativaL.)
Authors: Palevitz Barry A. , Mullinax J. Bennett 
Published: February 2005

AbstractChanges in microtubule organization were monitored in the stomatal complexes ofAvena sativausing tubulin immunocytochemistry. Radial arrays of cortical microtubules, previously thought to be characteristic of guard cells, also appear in adjacent subsidiary cells early in development. The subsidiary cell arrays are evident even before guard cells form via division of precursor guard mother cells. Thus, before the stomatal pore opens between sister guard cells, each complex contains four similar microtubule arrays. As the pore opens, however, the subsidiary cell system is reorganized into a network of microtubules distributed along the length of the cell. A similar change is effected in the guard cells after the pore opens. Subsidiary cells and guard cells elongate during later stages of differentiation, and a thickened wall is deposited int he narrow midzone of the latter. At the same time, microtubules in both cells assume a more axial orientation. The results are discussed in terms of developmental symmetry and the control of microtubule organization and cell wall deposition.
Altering the vector of polarity of BHK syncytia changes their motile behavior
Authors: Lewis‐Alberti Lindiann 
Published: February 2005

AbstractWe have previously shown that BHK syncytia have the ability to locomote provided the centrospheres are clustered and located adjacent to the cluster of nuclei. This article reports that experimental reorganizations of the centrospheres or the nuclei change the motile behavior of BHK syncytia in a way that is consistent with our previous observations: When fusion of the multiple nuclei occurred in stationary syncytia whose multiple nuclei encircled the centrosphere cluster, the centrospheres were expelled from the ring of nuclei. Consequently, locomotion was initiated in these syncytia even if they had been previously stationary for up to 5 days. Conversely, when a 2‐hour incubation in 5 μg/ml cytocholasin B caused the cluster of nuclei to surround the centrosphere cluster, the locomotion of the syncytia was inhibited. Similarly, the dispersal of the centrosphere cluster induced by a 4‐hour incubation in 1 μg/ml of colcemid resulted in the long‐term cessation of locomotion in motile syncytia.
Effects of calcium on motility of rainbow trout sperm flagella demembranated with triton X‐100
Authors: Okuno Makoto , Morisawa Masaaki 
Published: February 2005

AbstractSpermatozoa of the rainbow trout, Salmo gairdneri, were demembranated with Triton X‐100. The demembranated spermatozoa showed vigorous motility in the reactivation solution containing Ca2+ at the concentrations below 10−8.5M in the presence of cAMP. The motility was lost at 10−8M Ca2+ or more. The shape of the immotile flagella in the presence of high concentration of Ca2+ was not uniform: Some showed the cane shape and some were almost straight. The change in Ca2+ concentration of the extraction solution did not alter the motility of the reactivated spermatozoa. These results were different from those obtained from the sea urchin spermatozoa. When the concentration of cAMP was changed from 0.5 to 100 μM, the concentration of Ca2+ for converting the motile to immotile state was not altered. Thus, it is likely that the Ca2+‐dependent regulatory system of flagellar movement is independent of the cAMP‐induced initiation mechanism, which is assumed to require the transient influx of Ca2+ in rainbow trout spermatozoa.
Characterization of renatured profilin purified by urea elution from poly‐L‐proline agarose columns
Authors: Kaiser Donald A. , Goldschmidt‐Clermont Pascal J. , Levine Barry A. , Pollard Thomas D. 
Published: February 2005

AbstractWe present evidence that native profilin can be purified from cellular extracts of Acanthamoeba, Dictyostelium, and human platelets by affinity chromatography on poly‐L‐proline agarose. After applying cell extracts and washing the column with 3 M urea, homogeneous profilin is eluted by increasing the urea concentration to 6–8 M. Acanthamoeba profilin‐I and profilin‐II can subsequently be separated by cation exchange chromatography. The yield of Acanthamoeba profilin is twice that obtained by conventional methods. Several lines of evidence show that the profilins fully renature after removal of the urea by dialysis: (1) dialyzed Acanthamoeba and human profilins rebind quantitatively to poly‐L‐proline and bind to actin in the same way as native, conventionally purified profilin without urea treatment; (2) dialyzed profilins form 3‐D crystals under the same conditions as native profilins; (3) dialyzed Acanthamoeba profilin‐I has an NMR spectrum identical with that of native profilin‐I; and (4) dialyzed human and Acanthamoeba profilins inhibit actin polymerization. We report the discovery of profilin in Dictyostelium cell extracts using the same method. Based on these observations we conclude that urea elution from poly‐L‐proline agarose followed by renaturation will be generally useful for preparing profilins from a wide variety of cells. Perhaps also of general use is the finding that either myosin‐II or alpha‐actinin in crude cell extracts, can be bound selectively to the poly‐L‐proline agarose column depending on the ionic conditions used to equilibrate the column. We have purified myosin‐II from both Acanthamoeba and Dictyostelium cell extracts and alpha‐actinin from Acanthamoeba cell extracts in the appropriate buffers. These proteins are retained as complexes with actin by the agarose and not by a specific interaction with poly‐L‐proline. They can be eluted by dissociating the complexes with ATP and separated from actin by gel filtration if necessary.
Interaction between kinesin, microtubules, and microtubule‐associated protein 2
Authors: von Massow Alexander , Mandelkow Eva‐Maria , Mandelkow Eckhard 
Published: February 2005

AbstractKinesin from porcine brain was prepared by a procedure based on the strong binding of the protein to microtubules in the presence of sodium fluoride and ATP. The protocol reduces the requirement for taxol and AMP‐PNP. The kinesin is active in terms of its ability to move microtubules on glass slides and its ATPase. The ATPase of this kinesin is about 8 nmol/min/mg; it is activated to 19 nmol/min/mg in the presence of microtubules. The relationship between gliding velocity and ATP concentration follows Michaelis‐Menten kinetics. Using the motility assay, the maximal velocity is 0.78 μm/sec, and the Km values is 150 μM for ATP. For GTP the corresponding values are 0.38 μm/sec and 1.7 mM. ADP is a competitive inhibitor (Ki = 0.29 mM).Crude preparations of kinesin do not support motility on glass slides, whereas gel‐filtered kinesin does. A search for potential inhibitory factors showed that one of them is MAP2; however, its inhibitory effect becomes visible only in certain conditions. MAP2 bound to microtubules does not inhibit kinesin‐induced motility. However, when MAP2 and kinesin are preadsorbed to the glass surface independently of microtubules, MAP2 prevents the interaction of kinesin with microtubules, as if it formed a “lawn” that acted as a spacer and thus repelled the MAP‐free microtubules or crosslinked the MAP‐containing ones. The repelling effect of MAP2 domains (projection or assembly fragments obtained by chymotryptic cleavage) added separately is less pronounced and be overcome by kinesin. These results reinforce the view of MAP2 as a spacer molecule.
Assembly of the brush border cytoskeleton: Changes in the distribution of microvillar core proteins during enterocyte differentiation in adult chicken intestine
Authors: Heintzelman Matthew B. , Mooseker Mark S. 
Published: February 2005

AbstractThe assembly of the intestinal microvillus Cytoskeleton was examined during the differentiation of enterocytes along the crypt‐villus axis in adult chicken duodenum using light and electron microscopic immunolocalization techniques. Using antibodies reactive with villin, fimbrin, and the heavy chain (he) of brush border (BB) myosin I (110K‐calmodulin complex) and rhodamine‐conjugated phalloidin as a probe for F‐actin, we determined that while actin, villin, and fimbrin were all localized apically along the entire axis, BB myosin I (he) did not assume this localization until the crypt‐villus transition zone. In addition to their localization at the BB surface, all four proteins were present at significant levels along the lateral margins of enterocytes along the entire crypt‐villus axis, suggesting that these proteins may be involved in the organization and function of the basolateral membrane Cytoskeleton as well. The pattern of expression of the microvillar coreproteins along the crypt‐villus axis in the adult was comparable to that seen in the intestine of the late stage chicken embryo and suggests that a common program for brush border assembly may be used in both modes of enterocyte differentiation.
Macrophages form circular zones of very close apposition to lgG‐Coated surfaces
Authors: Heiple Jeanne M. , Wright Samuel D. , Allen Nina S. , Silverstein Samuel C. 
Published: February 2005

AbstractWhen phagocytes spread on surfaces coated with ligands such as IgG, they form a tight seal with the substrate. This seal excludes soluble macromolecules in the medium from the interface between the cell and substrate. In contrast, when cells spread on control surfaces that are not coated with ligands, the underside of the cell remains freely accessible to soluble proteins (Wright and Silverstein: Nature 309:359, 1984). We employed reflection‐interference microscopy (RIM) to determine where the seal forms during interaction with ligand (IgG) ‐coated surfaces. Human monocyte‐derived macrophages (MO) were plated at 37°C on dinitrophenylated (DNP)‐glass coverslips (control substrate), IgM anti‐DNP‐DNP‐coated glass (control substrate), or on IgG anti‐DNP‐DNP‐coated glass (phagocytosis‐promoting substrate). Live or fixed cells were examined by RIM. Spreading on control surfaces at 37°C was complete in 25 minutes, whereas spreading on IgG‐coated surfaces was maximal within 15 minutes and resulted in cell‐substrate contact area 1.6 × that of control cells. Within 1 h at 37°C, 90% of MO that spread on IgG‐coated substrates, but not on control substrates, excluded macromolecules from their underside. A minor population of cells (19%) exhibited a uniform iron gray RIM appearance indicating an even, close approach to the substrate. These cells may represent early stages of frustrated phagocytosis. In contrast to cells on control substrates, 70% of cells on IgG‐coated substrates developed continuous peripheral dark rings in RIM indicative of close association with the substrate. Essentially all cells with peripheral dark rings in RIM excluded macromolecules from their underside. Enclosed within this ring was an area of greater separation between the cell membrane and the substrate, as indicated by the lighter grey of this region in RIM and by the accessibility of substrate to anti‐substrate antibody when breaks in the dark ring occur. Thus, MO can create a closed compartment between plasma membrane and substrate that excludes proteins in the surrounding medium, thereby protecting substances secreted into this space from potentially inhibitory substances in the medium.
Outer‐arm dynein from trout spermatozoa: Substructural organization
Authors: King Stephen M. , Gatti Jean‐Luc , Moss Anthony G. , Witman George B. 
Published: February 2005

AbstractOuter‐arm dynein purified from trout spermatozoa was disrupted by low‐ionicstrength dialysis, and the resulting subunits were separated by sucrose density‐gradient centrifugation. The intact 19 S dynein, containing the α‐ an β‐heavy chains, intermediate chains (ICs) 1–5 and light chains (LCs) 1–6, yielded several discrete particles: a 17.5 S adenosine triphosphatase (ATPase) composed of the γ‐and β‐chains ICs 3–5 and LC 1; a 9.5 S complex containing ICs 1 and 2 together with LCs 2, 3, 4, and 6; and a single light chain (LC 5), which sedimented at ∼4 S. In some experiments, ICs 3–5 also separated from the heavy chain complex and were obtained as a distinct subunit. Further dissociation of the 17.5 S particle yielded a 13.1 S ATPase that contained the β‐heavy chain and ICs 3–5. The polypeptide compositions of the complexes provide new information on the intermolecular associations that occur within dynein.Substructural features of the trout dynein polypeptides also were examined. The heavy chains were subjected to vanadate‐mediated photolysis at the V1 sites by irradiation at 365 nm in the presence of Mg2+, ATP, and vanadate. Fragment pairs of relative molecular mass (Mr) 245,000/185,000 and 245,000/170,000 were obtained from the α‐ and β‐heavy chains, respectively. Photolysis of these molecules at their V2 sites, by irradiation in the presence of vanadate and Mn2+, yielded fragments of Mr 160,000/270,000 and 165,000/250,000, respectively. These values confirm that the α‐ and β‐heavy chains have masses of 430,000 and 415,000 daltons, respectively.Immunological analysis using monoclonal antibodies revealed that one intermediate chain from trout dynein (IC 2) contains epitopes present in two different intermediate chains from Chlamydomonas dynein. This indicates that specific sequences within the dynein intermediate chains have been highly conserved throughout evolution.
Direct observation of microtubule dynamics in Reticulomyxa: Unusually rapid length changes and microtubule sliding
Authors: Chen Yih‐Tai , Schliwa Manfred 
Published: February 2005

AbstractMicrotubule dynamics has been studied extensively in vitro, but comparatively little information is available on the in vivo behavior of microtubules. Here we report on the assembly, disassembly, and sliding of microtubules in the giant freshwater amoeba, Reticulomyxa. We have found that treating the cell with 0.25% trypsin induces the rapid formation of exceedingly flat areas within the reticulopodial network, allowing for the direct observation of microtubule behavior by DIC optics and computer‐enhanced video microscopy. In flattened areas, microtubule sliding occurs at rates of between 1 and 6.5 μm/sec. The average rate of microtubule assembly is 1.6 μm/sec, while microtubule disassembly takes place at about 4 μm/sec and can reach up to 19.5 μm/sec. We also observed many cases where a microtubule forms a hairpin loop and eventually breaks, resulting in bidirectional disassembly from the point of breakage. Our observations demonstrate sliding of cytoplasmic microtubules in vivo. The high rates of microtubule assembly/disassembly in this cell type are difficult to reconcile with conventional views of association and dissociation processes at microtubule ends and suggest unconventional mechanisms for the growth and shrinkage of microtubules.
Chromatin motion in neuronal interphase nuclei: Changes induced by disruption of intermediate filaments
Authors: Hay M. , De Boni U. 
Published: February 2005

AbstractMotion of nucleoli within interphase nuclei, known as nuclear rotation, may be used as a measure of motion of chromatin domains within the global confines of the nucleus. Mechanisms by which chromatin domains are transposed remain enigmatic. It has been established that nuclei are anchored by a network of intermediate filaments, structural proteins which share epitopes with nuclear lamins and possibly representing a constraint on nuclear rotation. It is postulated that selective removal of this constraint, by acrylamide, would result in increased chromatin motion. Mean rates of nucleolar displacement were quantified in neurons, in vitro. Nuclear rotation increased from a mean control rate of 0.102 ± 0.002 μm/min (n = 52) to a maximum mean rate of 0.207 ± 0.026 μm/min (n = 11), after 23 hr of exposure to 4 mM acrylamide. Despite this significant increase in motion of intranuclear domains, cytoplasmic structures in the immediate juxtanuclear area did not exhibit increases in rates of motion. Immunocy‐tochemistry was used to visualize cytoskeletal structures and to assay selective disruption of neurofilaments by acrylamide. Increased rates of chromatin motion coincided with breakdown of the intermediate filament network. Ultrastructural analyses showed that the increase in chromatin motion induced by acrylamide was also associated with a significant (P < 0.005) change in the thickness of the nuclear lamina, decreasing from 20.9 ± 5.10 nm (n = 159) in controls to 18.9 ± 3.1 nm (n = 148), to 19.5 ± 3.6 nm (n = 240) and to 16.1 ± 4.4 nm (n = 103) at 4, 8 and 22 hr exposure, respectively. Moreover, the number of mito‐chondria per unit area changed significantly (P < 0.0001) with exposure to acrylamide, increasing from 9.1 ± 2.2 mitochondrial profiles in controls to 16.5 ± 5.3 profiles after 22 hr exposure to acrylamide. Distribution of other cytoskel‐etal components, actin and microtubules, was not altered and does not appear to play a significant role in the observed increase in rates of nuclear rotation. We conclude that the removal of the damping effects on chromatin motion normally imposed by the nuclear lamina and by intermediate filaments results in increased chromatin motion.
Isolation and localization of a spectrin‐like protein from echinoderm sperm
Authors: D'Andrea Lisanne , Fishkind Douglas J. , Begg David A. , Bonder Edward M. 
Published: February 2005

AbstractThyone sperm undergo an explosive acrosome reaction resulting in the extension of a 90 μm long acrosomal process. In unreacted sperm, profilamentous actin is sequestered within the profilactin cup (Tilney: Journal of Cell Biology 69:73–89, 1976), which consists of four major polypeptides: actin, profilin, and a 250/235 kDa equimolar doublet (TS 250/235). Dialysis of profilactin preparations into an actin assembly buffer resulted in the formation of acrosomal‐like macromolecular aggregates containing actin, TS 250/235, and several other polypeptides as detected by SDS‐PAGE. TS 250/235 was purified by subjecting extracts of pH solubilized profilactin cups to DEAE and phosphocellulose ion exchange chromatography. TS 250/235 demonstrated immunocrossreactivity with affinity purified polyclonal antibodies raised against S. purpuratus egg spectrin. As determined by biotinylated‐calmodulin overlays, both subunits of TS 250/235 bound calmodulin in a Ca++‐sensitive manner. Electron microscopy of low angle, rotary shadowed replicas of TS 250/235 revealed an elongate rod‐shaped molecule with an average contour length of 203 nm. By indirect immunofluorescence, TS 250/235 was found to be uniformly distributed throughout the profilactin cup of the unreacted sperm. This distribution of TS 250/235 correlated with the location of monomeric actin as determined by localization studies utilizing fluorescent‐DNase‐1. Upon sperm activation, the cellular distribution of TS 250/235 dramatically changed and was observed both along the length and at the base of the extended acrosomal process.
Roles of actin filaments and three second‐messenger systems in short‐term regulation of chick dorsal root ganglion neurite outgrowth
Authors: Lankford Karen L. , Letourneau Paul C. 
Published: February 2005

AbstractIn a previous study (J. Cell Biol. 109:1229–1243, 1989), we reported that conditions which increased growth cone calcium levels and induced neurite retraction in cultured chick DRG neurons also resulted in an apparent loss of actin filaments in the growth cone periphery. We further showed that the actin‐stabilizing drug phalloidin could block or reverse calcium‐ionophore‐induced neurite retraction, indicating that the behavioral changes were mediated, at least in part, by changes in actin filament stability. In this study, we have further characterized the calcium sensitivity of growth cone behavior to identify which features of calcium‐induced behavioral effects can be attributed to effects on actin filaments alone, and to assess whether two other second‐messenger systems, cAMP and protein kinase C, might influence neurite outgrowth by altering calcium levels or actin stability. The results indicated that growth cone behavior was highly sensitive to small changes in calcium concentrations. Neurite outgrowth was only observed in calcium‐permeabilized cells when extracellular calcium concentrations were between 200 and 300 nM, and changes as small as 50 nM commonly produced detectable changes in behavior. Furthermore, low doses of cytochalasins mimicked all of the grossly observable features of growth cone responses to elevation of intracellular calcium, including the apparent preferential destruction of lamellipodial actin filaments and sparing of filopodial actin, suggesting that the behavioral effects of calcium elevation could be explained by loss of actin filaments alone. The effects of cAMP elevation and protein kinase C activation on growth cone behavior, ultrastructure, and fura2‐AM‐measured calcium levels indicated that the effects of cAMPmanipulations could be partially explained by a cAMP‐induced lowering of growth cone calcium levels and concomitant increased stabilization of actin filaments, but protein kinase C appeared to act through an independent mechanism.
Inhibition of intracellular granule movement by microinjection of monoclonal antibodies against caldesmon
Authors: Hegmann Theresa E. , Schulte Douglas L. , Lin Jenny Li‐Chun , Lin Jim Jung‐Ching 
Published: February 2005

AbstractMonoclonal antibodies, C2, C9, C18, and C21, against chicken gizzard caldesmon (called high molecular weight isoform) were shown to crossreact with a low molecular weight isoform of caldesmon in chicken embryo fibroblasts (CEF). These antibodies were used in a microinjection study to investigate the in vivo function of caldesmon in nonmuscle cell motility. Injected cells did not appear to change their morphology significantly; the cells displayed a flat appearance and were able to ruffle and locomote normally. However, in the C21 injected cells, saltatory movements of granules and organelles appeared to be greatly inhibited. This inhibition of granule movement was reversible, so that by 3 hr after injection, granules in injected cells had already recovered to normal speed. The inhibition of granule movement by C21 antibody was also very specific; the average speeds of granule movement in cells injected with C2, C9, or C18 antibody, or with C21 antibody preabsorbed with caldesmon, were not significantly different from that in uninjected cells. In a previous epitope study, we demonstrated that, of the antibodies used in this study, only C21 antibody was able to compete with the binding of caldesmon to Ca++/calmodulin and to F‐actin, although both C21 and C2 antibodies recognized the same carboxyl‐terminal 10K fragment of gizzard caldesmon [Lin et al., 1991: Cell Motil. Cytoskeleton 20:95–108]. The caldesmon distribution in C21 injected cells changed from stress‐fiber localization to a more diffuse appearance, when the injection was performed at 10–30 mg/ml of C21 antibody. We have previously shown that a monoclonal anti‐tropomyosin antibody exhibited motility‐dependent recognition of an epitope, and that micro‐injection of this antibody specifically inhibited intracellular granule movements of CEF cells [Hegmann et al., 1989: J. Cell Biol. 109:1141–1152]. Therefore, it is likely that tropomyosin and caldesmon may both function in intracellular granule movement by regulating the contractile system in response to [Ca++] change inside nonmuscle cells.