bluuebunny/crossref_metadata_2025 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

Changes 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

We 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

Spermatozoa 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

We 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

Kinesin 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

The 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

When 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

Outer‐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

Microtubule 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

Motion 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

Thyone 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

In 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

Monoclonal 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.
Inhibition of spindle elongation by taxol
Authors: Amin‐Hanjani S. , Wadsworth P.
Published: February 2005

During anaphase B spindle elongation, interzonal microtubules lengthen to accomplish pole‐pole separation, while at the same time remaining highly dynamic [Shelden and Wadsworth, J. Cell Sci. 97:273–281, 1990]. To further examine the role of microtubule polymerization and dynamics during spindle elongation, cells have been treated with taxol, which induces microtubule polymerization and stabilizes microtubules. Taxol was added to PtK1 cells 3 minutes after initial chromatid separation, so that the effect on anaphase B could be observed with minimal disruption to anaphase A movement. In 20 μM taxol, the rate and extent of pole‐pole separation, measured from time‐lapse video records, are reduced to 4% and 9.5% of controls, respectively. The organization of microtbules in taxol treated cells was examined using tubulin immunofluorescence and confocal fluorescence microscopy. Taxol induces a dramatic reorganization of interzonal microtubules resulting in a narrow gap, which is nearly completely lacking in MTs, across the center of the interzone. Furthermore, microtubules in taxol treated cells are resistant to nocodazole induced microtubule disassembly. Our results reveal that taxol rapidly inhibits anaphase B spindle elongation; inhibition is accompanied by a depletion of interdigitated interzonal microtubules and a reduction in microtubule dynamic behavior.
Kinky microtubules: Bending and breaking induced by fixation in vitro with glutaraldehyde and formaldehyde
Authors: Cross Alan R. , Williams Jr. Robley C.
Published: February 2005

We have employed video‐enhanced light microscopy to study alterations of the overall shape of microtubules that are produced by the aldehyde fixation methods commonly employed to study them in vitro. Changes brought about by these methods include deformation and breakage. The severity of the effects depends on the fixative employed and increases with its concentration, and with the time of fixation. The changes are observed under a variety of conditions, such as brief exposure to 3.7% formaldehyde, or somewhat longer exposure to glutaraldehyde at concentrations as low as 0.05%. The observed distortion explains why microtubules usually appear curved or sinuous in electron micrographs while appearing relatively rigid and linear in video‐enhanced light microscopy. The observed breakage implies that caution must be used in inferring length distributions from measurements of aldehyde‐fixed microtubules.
Cytoskeletal assembly and vinculin‐cytoskeleton interaction in different phases of the activation of bovine platelets
Authors: Horvath Andrea R. , Asijee Guus M. , Muszbek Laszlo
Published: February 2005

Vinculin is an Mr 130 kDa protein that has been implicated in membrane‐cytoskeleton interaction in various cell types. It has been demonstrated that vinculin is not a cytoskeletal component in resting platelets, but part of it becomes associated with the cytoskeleton during thrombin‐induced activation. In this study, using a quantitative immunnoblotting technique, the relation of vinculin to the cytoskeleton in different phases of activation of bovine platelets was explored, and the process of incorporation of vinculin into the cytoskeleton was related to that of cytoskeletal assembly. The assembly of cytoskeleton proceeded at a significantly faster rate than the association of vinculin with it, which shows that the latter process is not due to passive trapping of vinculin into the Triton‐insoluble residue, but certain biochemical changes had to occur before such an interaction became possible. When the formation of pseudopodia was prevented by cyto‐chalasin B, but neither aggregation nor the release reaction induced by thrombin were inhibited, the recovery of vinculin in the Triton‐insoluble residue even increased. In both time‐ and thrombin‐concentration‐dependent studies, poor correlation was found between vinculin‐cytoskeleton association and the extent of aggregation. Activation with phorbol‐myristate‐acetate, which is a strong stimulus for aggregation but produces only a slight release in the granular content, resulted in the association of only a negligible amount of vinculin with the cytoskeletal fraction. The incorporation of vinculin into the cytoskeletal fraction of thrombin activated platelets started with the release reaction but still proceeded, and the greatest part of the reaction occurred after secretion had gone to completion. These findings suggest that platelet shape change and pseudopodium extrusion are not prerequisites for, and aggregation is not related to, vinculin‐cytoskeleton interaction. The association of vinculin with the cytoskeleton correlates with the organization of contractile gel, which suggests a role for vinculin in secretion and clot retraction.
Differential sorting of beta tubulin isotypes into colchicine‐stable microtubules during neuronal and muscle differentiation of embryonal carcinoma cells
Authors: Falconer Marcia M. , Echeverri Christophe J. , Brown David L.
Published: February 2005

Pluripotent P19 embryonal carcinoma (EC) cells were differentiated along the neuronal and muscle pathways. Comparisons of class I, II, III, and IV beta tubulin isotypes in total and colchicine‐stable microtubule (MT) arrays from uncommitted EC, neuronal, and muscle cells were made by immunoblotting and by indirect immunofluorescence microscopy. In undifferentiated EC cells the relative amounts of these four isotypes are the same in both the total and stable MT populations. Subcellular sorting of beta tubulin isotypes was demonstrated in both neuronal and muscle differentiated cells. During neuronal differentiation, class II beta tubulin is preferentially incorporated into the colchicine‐stable MTs while class III beta tubulin is preferentially found in the colchicine‐labile MTs. The subcellular sorting of class II into stable MTs correlates with the increased staining of MAP IB. and with the expression of MAP 2C and tau. Although muscle differentiated cells express class II beta tubulin, stable MTs in these cells do not preferentially incorporate this isotype but instead show increased incorporation of class IV beta tubulin. Muscle cells do not show high levels of MAP IB and do not express MAP 2C or tau. These results are consistent with the hypothesis that a subcellular sorting of tubulin isotypes is the result of a complex interaction between tubulin isotypes and MT‐associated proteins.
Actin based motility on retraction fibers in mitotic PtK2 cells
Authors: Mitchison T. J.
Published: February 2005

When PtK2 cells round up in mitosis they leave retraction fibers attached between the substrate and the cell body. Retraction fibers and the region where they meet the cell body are rich in actin filaments as judged by phalloidin staining and electron microscopy. Video microscopy was used to study actin dependent motile processes on retraction fibers. Small, phase‐dense nodules form spontaneously on the fibers, and move in to the cell body at a rate of 3 μm/minute. As they move in they increase progressively in phase‐density. This movement appears to be related to actin dependent centripetal movement which has been previously studied in lamellipodia. Despite its generality, the mechanism of such movement is unknown, and retraction fibers present some special advantages for its study. Cytochalasin treatment causes nodules to stop moving and dissolve. Withdrawal of the drug causes them to reform and start moving. Surprisingly, movement after cytochalasin withdrawal was often outward, indicating a local reversal of cortical polarity. After a few minutes correct polarity is reestablished by a global control mechanism. The implications of these observations for the mechanism and polarity of actin dependent motility is discussed. © 1992 Wiley‐Liss, Inc.
Phalloidin binds and stabilizes pea root actin
Authors: Andersland John M. , Parthasarathy M. V.
Published: February 2005

Previous reports about phalloidin binding to plant actins have been indirect. We present here evidence showing that phalloidin does bind and stabilize filaments of actin extracted from pea roots. Criteria for the presence of actin included stabilization as a polymer in the presence of phalloidin, cross‐reaction with antibody against chicken actin, affinity binding to DNase I, and ability to be decorated by the S1 fragment of rabbit muscle myosin.

Phalloidin was able to stabilize polymers in pea root extracts against dissociation during SDS gel electrophoresis, and these polymers were shown to be composed exclusively of actin. Pea root actin isolated by affinity chromatography on a DNase I column was incubated with rhodamine phalloidin and electrophoresed on a native gel. The rhodamine fluorescence remained with the stabilized filaments, indicating clearly that phalloidin does bind to actin from a plant source. © 1992 Wiley‐Liss, Inc.
Desmosome assembly in MDCK epithelial cells does not require the presence of functional microtubules
Authors: Pasdar Manijeh , Li Zhi , Krzeminski Kathleen A.
Published: February 2005

Desmosomes, complex multisubunit structures that assemble at sites of cell‐cell contact, are important components of the epithelial junctional complex. Desmosome assembly requires the coordinated interaction at the plasma membrane of at least 8 cytoplasmic and integral membrane proteins organized into two structurally and functionally distinct domains, the cytoplasmic plaque and membrane core. Previous studies (Pasdar et al., J. Cell Biol., 113:645–655) provided evidence that cytokeratin filaments and microtubules may regulate transfer and assembly of cytoplasmic plaque and membrane core proteins, respectively. To determine directly the role of microtubules in these processes, Madin‐Darby canine kidney (MDCK) cells were treated with nocodazole or colchicine to disrupt the microtubular network. Biochemical analysis of the different components of the cytoplasmic plaque and membrane core domains revealed little or no effect of nocodazole or colchicine on the kinetics of synthesis, post‐translational modifications, transfer of proteins to the plasma membrane or their metabolic stability in the presence or absence of cell‐cell contact. Likewise, immunofluorescence analysis of desmosome formation demonstratedan apparently normal desmosome assembly in the presence of nocodazole or colchicine upon induction of cell‐cell contact. These results indicate that an intact microtubular network is not necessary for the processing or transport of the desmosomal membrane core glycoproteins to the plasma membrane in the absence or presence of cell‐cell contact. Furthermore, the integration of the cytoplasmic plaque and membrane core domains induced by cell‐cell contact at the plasma membranes of adjacent cells does not require the presence of functional microtubules. © 1992 Wiley‐Liss, Inc.
Tubulin dimer formation via the release of α‐ and β‐tubulin monomers from multimolecular complexes
Authors: Zabala Juan C. , Cowan Nicholas J.
Published: February 2005

The functional subunit of microtubules is a heterodimer consisting of α‐ and β‐tubulin. An understanding of tubulin dimerization has been hampered because it has not proved possible to purify native tubulin monomers. To study the process whereby tubulin dimers are formed, we made use of tubulins synthesized by in vitro transcription and translation. We present evidence that the in vitro synthesis of different mouse α‐tubulin isotypes involves a multimolecular complex. The synthesis of mouse β‐tubulin isotypes also involves the formation of multimolecular complexes, though different isotypes behave somewhat differently from one another. The properties of in vitro synthesized α‐ and β‐tubulin multimolecular complexes strongly suggest that they are intermediates in the biosynthesis of tubulin monomers. Upon release, these monomers can exchange with pre‐existing tubulin heterodimers. © 1992 Wiley‐Liss, Inc.
Characterization of smooth muscle caldesmon as a microtubule‐associated protein
Authors: Ishikawa Ryoki , Kagami Osamu , Hayashi Chihiro , Kohama Kazuhiro
Published: February 2005

We have previously shown that nonmuscle caldesmon copurified with brain microtubules binds to microtubules in vitro [Ishikawa et al.: FEBS Lett. 299:54–56, 1992]. To explore the role of caldesmon in the functions of microtubules, further characterization was performed using smooth muscle caldesmon, whose molecular structure and function have been best‐characterized in all caldesmon species.

Smooth muscle caldesmon bound to microtubules with a stoichiometry of five tubulin dimers to one molecule of caldesmon with the binding constant of 1.1 × 106M−1. The binding of caldesmon to microtubules was inhibited in the presence of Ca2+ and calmodulin. Partial digestion of the caldesmon with α‐chymotrypsin revealed that the binding site of the caldesmon for microtubules lay in the 34‐kDa C‐terminal domain. When the caldesmon was in the dimeric form in the absence of a reducing agent, the caldesmon cross‐linked microtubules to form bundles. Further, the caldesmon potentiated the polymerization of tubulin, and inhibited the in vitro movement of microtubules on dynein. These results suggest that caldesmon may be involved in the regulation by Ca2+ of the functions of microtubules. © 1992 Wiley‐Liss, Inc.
Thrombin, epidermal growth factor, and phorbol myristate acetate stimulate tubulin polymerization in quiescent cells: A potential link to mitogenesis
Authors: Ball Rebecca L. , Albrecht Thomas , Thompson William C. , James Olapade , Carney Darrell H.
Published: February 2005

Previous studies suggest that alterations in the microtubule (MT)‐tubulin equilibrium during G0/G1 affect mitogenesis. To determine the effect of growth factors on the MT‐tubulin equilibrium, we developed a radioactive monoclonal antibody binding assay (Ball et al.: J. Cell. Biol. 103:1033–1041, 1986). With this assay, 3H‐Ab 1 ‐ 1.1 binding to cytoskeletons in confluent populations of cultured cells is proportional to the number of tubulin subunits polymerized into MTs. We now show that purified α‐thrombin increases 3H‐Ab 1 ‐ 1.1 binding to cytoskeletons of serum‐arrested mouse embryo (ME) fibroblasts from 1.5‐ to 3‐fold. This stimulation is dose‐dependent and correlates with concentrations of thrombin required for initiation of DNA synthesis. Other mitogenic factors, epidermal growth factor (EGF) and phorbol 12‐myristate 13‐acetate (PMA), also stimulate MT polymerization. Addition of colchicine (0.3 μM) eight hours after growth factor addition blocks stimulation of 3H‐thymidine incorporation by thrombin, EGF, or PMA, suggesting that tubulin polymerization or subsequent events triggered by MT polymerization are required for cells to enter a proliferative cycle. Consistent with models for autoregulation of tubulin synthesis, thrombin, EGF, and PMA all increase tubulin synthesis 9 to 15 hr after growth factor addition, raising the possibility that the decrease in free tubulin and subsequent stimulation of tubulin synthesis is linked to progression of cells into a proliferative cycle. Colchicine addition to these cells also stimulates DNA synthesis, but colchicine‐stimulated cells enter S phase 6 to 8 hr later than those stimulated by growth factors. This delayed stimulation may be related to the time required for degradation of tubulin‐ colchicine complexes below a critical level. These data suggest that regulation of cell proliferation may be linked to increased MT polymerization and the resulting decrease in free tubulin pools. © 1992 Wiley‐Liss, Inc.
Native microtubules with a variable number of protofilaments
Authors: Dallai R. , Afzelius B. A. , Lanzavecchia S. , Bellon P. L.
Published: February 2005

Insect spermatozoa are characterized by having a set of accessory tubules that surrounds the microtubular doublets of the axoneme and that are formed from the B‐subtubules of the doublets. In trichopteran species, the accessory tubules have an unusually large diameter. Those of one species, Odontocerum albicorne, were seen to have a number of protofilaments that is 19 in the main part of the axoneme, but gradually decreasing to 18, 17, and 16 near the distal tip. The accessory tubule of the trichopteran axoneme has an asymmetrical shape and a skewed orientation, which makes it easy to distinguish a tubule that is viewed from its plus‐end from one viewed from the minus‐end. The shape of a cross‐sectioned protofilament in the trichopteran accessory tubules differs from that of microtubules in general, including accessory tubules of other insects, by being polygonal with the most acute angle pointing centripetally. © 1993 Wiley‐Liss, Inc.
Molecular cloning and expression of the chicken smooth muscle γ‐actin mRNA
Authors: Kovacs Adrienne M. , Zimmer Warren E.
Published: February 2005

We have investigated the expression of chicken smooth muscle γ‐actin mRNA by isolation and characterization of cDNAs representing this actin isoform and utilizing the cDNA to probe RNA from adult and developing cells. Nucleotide sequence elucidated from an apparent full length smooth muscle γ‐actin cDNA revealed that it contained 94 bp of 5′ non‐translated sequence, an open reading frame of 1131 bp, and 97 bp of 3′ non‐translated sequence. Within the 376 amino acid sequence deduced from the chicken cDNA were diagnostic amino acids at the NH2‐ and COOH‐terminal regions which provided unequivocal identification of the γ‐enteric smooth muscle actin isoform. In addition, the chicken γ‐enteric actin deduced from our cDNA clones was found to differ from the sequence reported in earlier protein studies [J. Vandekerckhove and K. Weber, FEBS Lett. 102:219, 1979] by containing a proline rather than a glutamine at position 359 of the protein, indicating that the avian γ‐enteric actin isoform is identical to its mammalian counterpart. Comparison of the 5′ and 3′ non‐translated sequence determined from the chicken cDNA to that elucidated for rat, mouse, and human showed that there is not a high degree of cross‐species sequence conservation outside of the coding regions among these mRNAs. Northern hybridization analyses demonstrated that the γ‐enteric actin mRNA is expressed in adult aorta and oviduct tissues but not in adult skeletal muscle, cardiac muscle, liver, brain, and spleen tissues. The γ‐enteric actin mRNA was first observed in measurable quantities in gizzard tissue from 4–5 day embryos and increased in content in developing smooth muscle cells through 16–17 embryonic days. Following this initial increase during embryonic development, the γ‐enteric actin mRNA exhibits a decline in content until ∼7 days posthatching, after which there is an increase in content to maximal levels found in adult gizzard tissue. In general, the developmental appearance of the γ‐enteric mRNA parallels that observed for this protein in previous studies indicating that the developmental expression of smooth muscle γ‐actin is regulated, in part, by an increased content of mRNA in chicken visceral smooth muscle cells during myogenesis. © 1993 Wiley‐Liss, Inc.
Incorporation of microinjected mutant and wildtype recombinant tropomyosins into stress fibers in fibroblasts
Authors: Ranucci Denise , Yamakita Yoshihiko , Matsumura Fumio , Hitchcock‐DeGregori Sarah E.
Published: February 2005

The structural requirements for assembly of tropomyosin into stress fibers were investigated by microinjecting wildtype and four mutant striated chicken muscle α‐tropomyosins expressed in E. coli as fusion and nonfusion proteins into cultured rat embryo fibroblasts, followed by localization of tropomyosin using indirect immunofluorescence. The results show that the determinants for stress fiber incorporation in living cells correlate with the in vitro actin affinity of these tropomyosins. Wildtype recombinant protein incorporated into stress fibers both when the amino terminus was unacetylated and when it was blocked with an 80‐residue fusion protein [Hitchcock‐DeGregori, S.E., and Heald, R.W. (1987): J. Biol. Chem. 262:9730–9735]. The pattern of incorporation was indistinguishable from that of tropomyosin isolated from chicken pectoral muscle. The striated α‐tropomyosin incorporated into stress fibers, even though this isoform is not found in nonmuscle cells. Three recombinant mutant tropomyosins in which one‐half, two‐thirds, or one actin binding site was deleted were tested [Hitchcock‐DeGregori, S.E., and Varnell, T.A. (1990): J. Mol. Biol. 214:885–896]. Only the fusion protein with a full actin binding site deleted incorporated into stress fibers. However, the unacetylated, nonfusion proteins with one half and one actin binding site deleted incorporated into stress fibers, consistent with the ability of troponin to promote the actin binding in vitro. A fourth mutant, in which the conserved amino‐terminal nine residues were deleted, did not incorporate into stress fibers, consistent with the complete loss of function of this mutant [Cho, Y.J., Liu, J., and Hitchcock‐DeGregori, S.E. (1990): J. Biol. Chem. 265:538–545]. © 1993 Wiley‐Liss, Inc.
Effects of taxol and taxol/hyperthermia treatments on the functional polarization of cytotoxic T lymphocytes
Authors: Knox J. David , Mitchel Ron E. J. , Brown David L.
Published: February 2005

Immunofluorescence staining, electron microscopy, and (51Cr) cytolytic release assays are used to investigate the effects of taxol and taxol/hyperthermia treatments on the microtubule organization and cytolytic activity of cytotoxic T lymphocytes (CTLs). A 4 h treatment of CTLs with 1 μM taxol results in an extensive reorganization of the microtubule system to form one to a few large microtubule bundles that extend from the centrosome. The Golgi apparatus is not disrupted by this treatment and remains associated with the microtubule organizing centre (MTOC). This microtubule reorganization has no effect on the ability of CTLs to orient their MTOC towards a bound target cell, nor on their cytolytic activity. In control CTLs, not treated with taxol, a mild hyperthermia treatment (42°C, 30 min) results in an aggregation of the pericentriolar material, a loss of MTOC orientation, an inhibition of cytolytic activity, and a disorganization of the microtubule system [Knox et al.: Exp. Cell Res. 194:275–283, 1991]. In contrast, in taxol‐treated CTLs the stabilized microtubule bundles are unaffected by such hyperthermia treatment; however, the other effects of hyperthermia appear identical in control and taxol‐treated CTLs. These results indicate that a dynamic, radially arranged microtubule array is not required for the functional polarization of CTLs and suggest that a component of the pericentriolar material may play a key role in effecting MTOC orientation. © 1993 Wiley‐Liss, Inc.
Ultrastructure and immunocytochemistry of the isolated human erythrocyte membrane skeleton
Authors: Ursitti Jeanine A. , Wade James B.
Published: February 2005

Isolated skeletons from human erythrocyte ghosts were studied using immunogold labeling; negative staining; and quick‐freeze, deep‐etch, rotary replication with Pt/C (QFDERR). Isolated skeletons visualized by QFDERR were similar to the negatively stained skeletons in that the proteins spectrin, actin, and ankyrin could be easily distinguished. However, the quick‐frozen skeletons had two fewer filaments (4.2 ± 0.7) at an actin junction. Immunogold labeling of skeletons with site‐specific spectrin antibodies not only confirmed the designation of these filaments as spectrin molecules, but indicated that about 30% of spectrin filaments form non‐actin junctions consistent with the hexameric organization of these filaments. Many of the filaments displayed a striking banding pattern indicative of underlying substructure. Isolated skeletons prepared by QFDERR also showed evidence of laterally associated spectrin filaments. These associations, as well as many hexamer junctions, are lost during negative staining. Negative staining also apparently caused ∼21% of the spectrin filaments to separate into their monomeric subunits. These results indicate that the surface tension imposed during negative staining of isolated skeletons can cause a loss of interactions normally present in the intact membrane skeleton. © 1993 Wiley‐Liss, Inc.