bluuebunny/crossref_metadata_2025_split huggingface dataset:
Buckling of a single microtubule by optical trapping forces: Direct measurement of microtubule rigidity
Authors: Kurachi Masashi , Hoshi Masayuki , Tashiro Hideo 
Published: February 2005

AbstractAs major determinants of cell shape and polarity, microtubules are required to have suitable rigidity. However, our knowledge of the mechanical properties of microtubules is far from satisfactory. We report here a new method of measuring the flexural rigidity of a single microtubule by direct buckling using the optical trapping technique. Microtubule buckling was induced by applying a small longitudinal compressing force through an optically trapped microsphere that was firmly attached to the microtubule. Three ways of estimating the flexural rigidity of a continuous slender rod, one from the observed critical load of buckling and two from deflected lengths and angles of bending, yielded values which agreed well when applied to the analysis of buckling microtubules. Unexpectedly, we found that the rigidity was not constant as expected but was dependent on microtubule length. This length dependency explains the discrepancies among reported values of microtubule flexural rigidity measured by different methods. Comparing microtubules of identical lengths, microtubules assembled with brain‐derived associated proteins (4 × 10−23 Nm2 at around 10 m̈m in length) were four times more rigid than those assembled from purified tubulin and stabilized with taxol (1 × 10−23 Nm2). © 1995 Wiley‐Liss, Inc.
Protein substrates for CGMP‐dependent protein phosphorylation in cilia of wild type and atalanta mutants of Paramecium
Authors: Ann Kyoung‐Sook , Nelson David L. 
Published: February 2005

AbstractIn the ciliated protozoan Paramecium, swimming direction is regulated by voltage‐gated Ca2+ channels in the ciliary membrane. In response to depolarizing stimuli, intraciliary Ca2+ rises, triggering reversal of the ciliary power stroke and backward swimming. One class of Ca2+ ‐unresponsive behavioral mutants of Paramecium, atalanta mutants, cannot swim backward even though they have functional Ca2+ channels in their ciliary membrane. Several atalanta mutants were characterized with regard to several Ca2+ ‐dependent activities, but no significant difference between wild type and the mutants was detected. However, one allelic group, atalanta A (initially characterized by Hinrichsen and Kung [1984: Genet. Res. Camb. 43:11–20]), showed a helical swimming path of opposite handedness from that of wild‐type cells when detergent‐permeabilized cells (“models”) were reactivated with MgATP. When cGMP‐dependent protein kinase purified from wild‐type cells was added to atalanta A models, the handedness of the swimming path was reversed. Cyclic GMP stimulated in vitro phosphorylation of several proteins in isolated cilia, and the pattern of phosphoproteins was very similar for wild type and atalanta mutants, with one exception: a protein of 59 kDa was phosphorylated much less in the mutant ata A. When ciliary proteins were separated by gel electrophoresis and then phosphorylated “on blot” by purified cGMP‐dependent protein kinase, phosphoprotein patterns were similar in wild type and ata mutants except that a 48 kDa protein (p48) from ata A3 was more heavily phosphorylated. This difference in p48 phosphorylation was also observed with cGMP‐dependent protein kinase purified from ata A3 mutant cells. Ciliary p48 may be part of the mechanism that regulates the orientation of the ciliary power stroke. © 1995 Wiley‐Liss, Inc.
Differentiation dependent expression of tensin and cortactin in chicken osteoclasts
Authors: Hiura Kenji , Lim Soo‐Siang , Little Sheila P. , Lin Shin , Sato Masahiko 
Published: February 2005

AbstractThe expression and localization of tensin and cortactin were examined in osteoclast precursors in comparison with isolated osteoclasts on various substrates. Initially, the ability of hen monocytes to differentiate into osteoclasts was evaluated on plastic or glass, and compared to differentiation on bone. Specifically, monocytes were isolated from the medullary bones of egg‐laying hens maintained on a Ca‐deficient diet. Differentiation was monitored morphologically and by quantitation of the ability to form Howship's lacunae in bone slices or resorb radiolabeled bone particles of 20–53 m̈m diameter. These cells differentiated into tartrate resistant acid phosphatase (TRAP)‐positive, bone resorbing, multinucleated syncytia in the presence of cytosine‐1‐β‐D‐arabinofuranoside in a time dependent manner (day 1–6). Differentiation into osteoclast‐like cells was similar whether cultured on plastic, on glass, or on bone. When compared to GAP‐DH control levels, tensin and cortctin mRNA levels increased by 7‐ and 10‐fold, respectively, by day 6. Tensin and cortactin protein levels also increased by 6‐ and 15‐fold, respectively, by day 6. Immunofluorescence of differentiating precursors showed that tensin localized between regions of cell to cell contact and colocalized with vinculin in podosomes of osteoclast‐like cells and of real osteoclasts. Cortactin immunofluorescence was not detectable in monocytes but localized inside tensin/vinculin podosome structures after fusion into osteoclast‐like cells and in freshly isolated osteoclasts. Both tensin and cortactin were associated with attachment complexes used by osteoclast‐like cells and osteoclasts to resorb bone. Specifically, punctate cortactin staining was observed inside tensin staining which formed a double ring structure at the membrane/bone interface of resorbing osteoclasts. These data showed that tensin and cortactin can be used as osteoclast differentiation markers, that participate in attachment complexes used to resorb bone, and that tensin may participate in the fusion process of osteoclast precursors. © 1995 Wiley‐Liss, Inc.
Polylysine cross‐links axoplasmic neurofilaments into tight bundles
Authors: Brown Anthony , Lasek Raymond J. 
Published: February 2005

AbstractWe have used axoplasm from the squid giant axon to investigate the effects of anionic and cationic polypeptides on the mobility and organization of axonal neurofilaments (NFs). Intact cylinders of axoplasm were extruded from squid giant axons into an excess volume of artificial axoplasm solution. In a previous study on the mobility of NFs in extruded axoplasm, we showed that these polymers disperse freely and diffusively into the surrounding solution, thereby expanding the axoplasmic cross‐sectional area [Brown and Lasek, 1993: Cell Motil. Cytoskeleton 26:313–324]. In the present study, we found that 83nm‐long (“long‐chain”) polylysine, a synthetic multivalent cationic protein, inhibited the radial expansion of isolated axoplasm and condensed the axoplasm, thereby reducing the cross‐sectional area. Equivalent concentrations of a 7nm‐long (“short‐chain”) polylysine did not inhibit the expansion of axoplasm and did not cause the axoplasm to condense. Inhibition of the expansion of axoplasm by long‐chain polylysine was dependent on the polylysine concentration; condensation of axoplasm was observed at concentrations of 0.01 mg/ml (0.27 μM) or greater. Electron microscopy of the condensed axoplasm showed that the NFs were aligned side‐by‐side and in parallel in closely‐packed bundles. Equivalent concentrations of 91nm‐long (“long‐chain”) polyglutamate, a synthetic multivalent anionic protein, partially inhibited the expansion of axoplasm but did not cause the NFs to bundle and did not cause the axoplasm to condense. These studies indicate that cationic proteins bind tightly to the highly charged anionic surfaces of NFs and can link them together into compact bundles in a charge‐dependent and length‐dependent manner. The tightly packed organization of these cross‐linked NFs differs from the normal loose organization of NFs in healthy axons. However, tightly bundled NFs are sometimes found in certain neuropathologies, such as giant axonal neuropathy.
Outer arm dynein from newt lung respiratory cilia: Purification and polypeptide composition
Authors: Rupp Gerald , Hard Robert 
Published: February 2005

AbstractDyneins are multimeric ATPases that comprise the inner and outer arms of cilia and flagella. It previously has been shown that salt extraction of newt lung axonemes selectively removes >95% of the outer arm dynein (OAD), and that the beat frequency of OAD‐depleted axonemes cannot be activated as compared to controls [Hard et al., 1992: Cell Motil. Cytoskeleton 21:199–209]. Therefore, expression of the activated state appears to require the presence of outer dynein arms. The presen study was undertaken to ascertain basic information on the structure and molecular composition of newt OAD. Populations of demembranated axonemes were extracted with 0.375 M salt. Each lung released ∼ 1.4 × 107 axonemes during isolation, yielding ∼ 120 ng of salt extractable OAD. Electron microscopy of negatively stained samples revealed that newt OAD consisted of two globular heads joined together by a Y‐shaped stem, similar to sea urchin and trout sperm OAD. Each head appeared to be roughly spherical in shape, measuring ∼ 17 nm in diameter. Electrophoretic analysis of whole axonemes revealed more than six dynein heavy chains when resolved in silver stained 0–8 M urea, 3–5% acrylamide gradients. Extracted OAD, either crude in high salt or purified by alloaffinity, was composed of two heavy chains. UV‐induced (366 nm) photolytic cleavage at the V1 site, performed in the presence of Mg2+, vanadate, and ATP, produced four new polypeptides (Mr 234, 232, 197, and 189 kD). Photolysis was supported by Mg2+ and Ca2+, but did not occur in the presence of Mn2+. The apparent Mr of the dynein heavy chains was determined to lie between 430–420 kD. Eight discrete polypeptides (putative intermediate chains, IC1‐IC8, Mr 175–56 kD) copurified with the α‐ and β‐heavy chains by microtubule‐alloaffinity.Based on its extraction characteristics, polypeptide composition in purified and crude samples, and structure, we conclude that this two‐headed particle represents the entire newt respiratory outer arm dynein.
Focal adhesion formation is associated with secretion of allergic mediators
Authors: Kawasugi Kazuo , French Peter W. , Penny Ronald , Ludowyke Russell I. 
Published: February 2005

AbstractAdherence of cells to the extracellular matrix via focal adhesions is known to modulate many cellular functions. However, the role of focal adhesions in the regulation of secretion is unclear. To examine this we have used the RBL‐2H3 rat mast cell line, in which we and others have observed cytoskeletal rearrangements and increased cell spreading during secretion. All activators of secretion examined, whether acting specifically through or bypassing the IgE‐receptor, induced the assembly of focal adhesions, as defined by the localization of vinculin and talin. The extent of focal adhesion formation correlated with the extent of secretion and the time course of secretion also correlated with that of the assembly of focal adhesions. To examine the mechanism by which focal adhesion formation occurred, the protein kinase C inhibitor bisindolylmaleimide was used. Bisin‐dolylmaleimide caused complete inhibition of both secretion and focal adhesion formation induced by antigen or the calcium ionophore A23187. Although PMA did not induce secretion, it induced focal adhesion assembly which was inhibited by bisindolylmaleimide. The inhibitor had no effect on secretion or focal adhesion formation induced by the ATP analogue, ATPγS in permeabilized cells, indicating ATPγS acts after the activation of protein kinase C in the secretory pathway. These data provide novel evidence that the formation of focal adhesions may have a role in the process of secretion from mast cells.
In ascaris sperm pseudopods, msp fibers move proximally at a constant rate regardless of the forward rate of cellular translocation
Authors: Royal Dewey , Royal Maryanne , Soll David R. , Italiano Joseph , Roberts Tom 
Published: February 2005

AbstractComputer‐assisted methods have been employed to obtain a high resolution description of pseudopod expansion, cellular translocation, and the subcellular dynamics of MSP fiber complexes in the motile sperm of the nematode Ascaris suum. Although Ascaris sperm translocating in a straight line or along a curved path do not retract their pseudopod or significantly alter pseudopod shape, they move in a cyclic fashion, with an average period between velocity peaks of 0.35 × 0.05 min, which is independent of the forward velocity of sperm translocation. Expansion is confined to a central zone at the distal edge of the pseudopod for sperm translocating in a straight line and to a left‐handed or right‐handed lateral zone in the direction of turning, for sperm translocating along a curved path. For cells translocating in a straight line, the branch points and kinks of MSP fiber complexes move in a retrograde direction in relation to the substratum at an average velocity of 11 μm per min which is independent of the forward velocity of sperm translocation. The distal (anterior) end of a fiber complex, however, moves distally at the speed of sperm translocation when it emanates from the expansion zone, but when it is displaced to a nonexpanding surface of the pseudopod, it stops moving distally. When a cell is anchored to the substratum and is, therefore, nonmotile, the velocity of fiber complexes moving in a retrograde direction doubles. The unique aspects of pseudopod and MSP fiber complex dynamics in Ascaris are compared to the dynamics of pseudopod formation and actin filament dynamics in traditional actin‐based amoeboid cells, and the treadmill model for MSP polymerization is reassessed in light of the discovery that fiber complex branch points move proximally (posteriorly) at a fixed rate.
Increasing intracellular concentrations of thymosin β4 in PtK2 cells: Effects on stress fibers, cytokinesis, and cell spreading
Authors: Sanger Jean M. , Golla Rajasree , Safer Daniel , Choi John K. , Yu Keh‐Ru , Sanger Joseph W. , Nachmias Vivianne T. 
Published: February 2005

AbstractThymosin β4 (Tβ4) binds to G‐actin in vitro and inhibits actin polymerization. We studied the effects of incresing Tβ4 concentration within living PtK2 cells, comparing its effects on the disassembly of stress fibers and membrane‐associated actin with its ability to inhibit cytokinesis and cell spreading after mitosis. We chose PtK2 cells for the study because these cells have many striking actin bundles in both stress fibers and cleavage furrows. They also have prominent concentrations of membrane‐associated actin and remain flattened during mitosis. We have found that PtK2 cells contain an endogenous homologue of Tβ4 at a concentration (approximately 28 μM) sufficient to complex a third or more of the cell's unpolymerized actin. Intracellular Tβ4 concentrations were increased by three different methods: (1) microinjection of an RSV vector containing a cDNA for Tβ4; (2) transfection with the same vector; and (3) microinjection of purified Tβ4 protein. The plasmid coding for Tβ4 was microinjected into PtK2 cells together with fluorescently labeled alpha‐actinin as a reporter molecule. Immediately after microinjection fluorescently labeled alpha‐actinin was detected in a periodic pattern along the stress fibers just as in control cells injected solely with the reporter. However, after 13 h, cells microinjected with reporter and plasmid showed marked disassembly of the fiber bundles. PtK2 cells transfected with this RSV vector for 2–3 days showed disassembly of stress fibers as detected by rhodamine‐phalloidin staining; in these cells the membrane actin was also greatly diminished or absent and the border of the cells was markedly retracted. Microinjection of pure Tβ4 protein into interphase PtK2 cells induced disassembly of the stress fibers within 10 min, while membrane actin appeared only somewhat reduced. If the PtK2 cells were mitotic, Similar microinjection of pure thymosin β4 protein at times from early prophase to metaphase resulted in an unusual pattern of delayed cytokinesis. Furrowing occurred but at a much slower rate than in controls and the amount of actin in the cleavage furrow was greatly reduced. The cells constricted to apparent completion, but after about 30 min the furrow re‐gressed, forming a binucleate cell, much as after treatment with cytochalasin B or D. Postcytokinesis spreading of these Tβ4‐injected cells was often inhibited. These experiments suggest that an insufficient number of actin filaments prolongs the contractile phase of cytokinesis and abolishes the final sealing process. © 1995 Wiley‐Liss, Inc.
Characterization of adhesion plates induced by the interaction of Entamoeba histolytica trophozoites with fibronectin
Authors: Váazquez Joséa , Franco Elizabeth , Reyes Guadalupe , Meza Isaura 
Published: February 2005

AbstractEntamoeba histolytica trophozoites are pleiomorphic and highly motile cells. Although scarce fibrous material can be identified in the cytoplasm as elements of an organized cytoskeleton, clearly defined actin‐containing structures are formed at the sites of cell‐matrix contact upon the interaction of trophozoites with fibronectin (FN) and other extracellular matrix substrates. These structures are reminiscent of the adhesion plaques or focal contacts found in higher eukaryotic cells, where actin filament bundles insert into specialized regions of the plasma membrane and function as signal transduction organelles. Thus, the formation of adhesion plates in this parasitic ameba could be related to specific signaling responses involved in its invasive behavior. Here, we report the isolation of amebic adhesion plates and the results of their structural and molecular analyses. Filaments, with the characteristic diameter of F‐actin, radiating from an electrondense matrix, are the main feature. Actin is one of the main protein components of the plate; other proteins identified are a FN‐binding protein—previously reported as a “putative” FN receptor—the actin‐binding proteins myosin II, myosin I, α‐actinin, vinculin, and tropomyosin. The presence in the isolated plates of several proteases and protein kinases, in particular pp125FAK, is also demonstrated. Our results suggest that adhesion plates in amebas are dynamic membrane‐cytoskeletal complexes participating not only in the attachment to FN substrates but also providing the structural basis for their involvement in parasite locomotion and invasiveness. © 1995 Wiley‐Liss, Inc.
Cytochalasin J affects chromosome congression and spindle microtubule organization in PtK1 cells
Authors: Snyder Judith A. , Cohen Laura 
Published: February 2005

AbstractPtK1 cells were treated with 10 μg/ml cytochalasin J (CJ) for 15 min at various stages of mitosis. When applied at nuclear envelope breakdown (NEB) chromosome congression was blocked or substantially slowed, and chromosomes failed to show organization patterns typical of prometaphase. Spindle microtubule (MT) numbers appeared unaffected as judged by the pattern of birefringent retardation. However, ultrastructural analysis showed MTs to be reorganized within the spindle domain with some exhibiting fragmentation and others failing to interact with poorly defined kinetochore laminae. The spindle domain took on a curved, almost banana‐like shape, as related to the position of the centrosomes and lack of orientation of chromosomes. Serial section analysis of kinetochore regions showed reduced contour length and maturation of the kinetochore plate with few MTs associated with this structure. Cells similarly treated with 10 μg/ml CJ at NEB for 15 min and then released into conditioned medium for 15 min showed that most chromosomes resumed congression to the metaphase plate. Ultrastructural analysis revelaed a more normal organization of spindle MTs, but kinetochore structure remained affected. CJ treatment of cells in prometaphase slightly affected chromosome congression with most chromosomes aligning at the metaphase plate after 10–15 min of treatment. Ultrastructural analysis showed that astral MTs were disrupted and spindle MTs were fragmented; few MTs coursed from kinetochore to pole. Kinetochore structure was also affected with only small numbers of short MTs seen associated with kinetochores. Application of CJ at anaphase onset had little effect on anaphase A and B, but cytokinesis failed to occur. Anti‐tubulin staining of a monolayer of cells treated with 10 μg/ml CJ for 15 min showed that over 60% of mitotic figures exhibited changes in MT organization. Cells showing the greatest effect of treatment had several foci of bundles of MTs, as if the spindle were multipolar. Chromosomes were arranged near the periphery of the spindle which could be a result of abnormalities of kinetochore structure. Improper association of spindle MTs with kinetochores and, thus, changes in kinetochore position could account for these changes in spindle architecture. © 1995 Wiley‐Liss, Inc.
Equilibrium and growth shapes of crystals: how do they differ and why should we care?
Authors: Sekerka Robert F. 
Published: February 2005

AbstractSince the death of Prof. Dr. Jan Czochralski nearly 50 years ago, crystals grown by the Czochralski method have increased remarkably in size and perfection, resulting today in the industrial production of silicon crystals about 30 cm in diameter and two meters in length. The Czochralski method is of great technological and economic importance for semiconductors and optical crystals. Over this same time period, there have been equally dramatic improvements in our theoretical understanding of crystal growth morphology. Today we can compute complex crystal growth shapes from robust models that reproduce most of the features and phenomena observed experimentally. We should care about this because it is likely to result in the development of powerful and economical design tools to enable future progress. Crystal growth morphology results from an interplay of crystallographic anisotropy and growth kinetics by means of interfacial processes and long‐range transport. The equilibrium shape of a crystal results from minimizing its anisotropic surface free energy under the constraint of constant volume; it is given by the classical Wulff construction but can also be represented by an analytical formula based on the ξ‐vector formalism of Hoffman and Cahn. We now have analytic criteria for missing orientations (sharp corners or edges) on the equilibrium shape, both in two (classical) and three (new) dimensions. Crystals that grow under the control of interfacial kinetic processes tend asymptotically toward a “kinetic Wulff shape”, the analogue of the Wulff shape, except it is based on the anisotropic interfacial kinetic coefficient. If it were not for long range transport, crystals would presumably nucleate with their equilibrium shape and then evolve toward their “kinetic Wulff shape”. Allowing for long range transport leads to morphological instabilities on the scale of the geometric mean of a transport length (typically a diffusivity divided by the growth speed) and a capillary length (of the order of atomic dimensions). Resulting crystal growth shapes can be cellular or dendritic, but can also exhibit corners and facets related to the underlying crystallographic anisotropy. Within the last decade, powerful phase field models, based on a diffuse interface, have been used to treat simultaneously all of the above phenomena. Computed morphologies can exhibit cells, dendrites and facets, and the geometry of isotherms and isoconcentrates can also be determined. Results of such computations are illustrated in both two and three dimensions. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)