bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of cytochalasin and colcemid on cortical flow in coelomocytes
Authors: Edds Kenneth T. 
Published: February 2005

AbstractSea urchin coelomocytes naturally flatten on a substratum into a discoid morphology and display striking, centripetally directed cortical flow along the radii of the cell when viewed with time lapse, video enhanced microscopy. The rate of cortical flow averaged 4.5 μm/min in the peripheral most 10 μm of cytoplasm but slows considerably in the perinuclear region. Cytochalasin B causes: (1) the flow to stop, (2) the buildup of an actin filament‐rich peripheral ridge of cytoskeletal material, (3) the centrifugal dissolution of a portion of the actin cytoskeleton, and (4) the contraction of other portions of the cytoskeleton into foci. Cytochalasin D (CD), on the other hand, causes the flowing actin meshwork to become severed from the edge of the cell and allows it to be drawn at least part way in towards the nucleus. A smaller peripheral ridge of actin filament buildup is also seen with CD. Colcemid induces another striking change in the cytoskeleton. The centripetal progression of the actin is not stopped by colcemid, but shortly after leaving the periphery of the cell, the linear elements within the flow become reoriented into arcs. The long axis of the arcs is roughly parallel with the cell's edge. The effects of all three drugs are reversible. The results are discussed in light of other systems and potential mechanisms for cortical flow. © 1993 Wiley‐Liss, Inc.
Contractile protein dynamics of myofibrils in paired adult rat cardiomyocytes
Authors: Imanaka‐Yoshida Kyoko , Sanger Jean M. , Sanger Joseph W. 
Published: February 2005

AbstractThe purpose of this study was to determine how quickly contractile proteins are incorporated into the myofibrils of freshly isolated cardiomyocytes and to determine whether there are regions of the cells that are more dynamic than others in their ability to incorporate the proteins. Paired cardiomyocytes joined at intercalated discs and single cells were isolated from adult rats, and microinjected 3 hours later with fluorescently labeied actin, alpha‐actinin, myosin light chains, and vinculin. The cells were fixed and permeabilized at various period, 5 seconds and longer, after microinjection. Actin became incorporated throughout the I‐Bands in as short a time as 5 seconds. The free edges of the cells, which were formerly intercalated discs, exhibited concentrations of actin greater than that incorporated in the I‐Bands. This extra concentration of actin was not detected, however, at intact intercalated discs connecting paired cells. Alpha‐actinin was incorporated immediately into Z‐Bands and intercalated discs. Vinculin, also, was localized at the Z‐Bands and at intercalated discs, but in contrast to alpha‐actinin, there was a higher concentration of vinculin in the region of the intact intercalated discs. Both alpha‐actinin and vinculin were concentrated at the free ends of the cells that were formerly parts of intercalated discs. Myosin light chains were observed to incorporate into the A‐Bands in periods as short as 5 seconds. These results suggest that the myofibrils of adult cardiomyocytes may be capable of rapid isoform transitions along the length of the myofibrils. The rapid accumulation of fluorescent actin, alpha‐actinin, and vinculin in membrane sites that were previously parts of intercalated discs, may reflect the response to locomotory activity that is initiated in these areas as cells spread in culture. A similar response after an injury in the intact heart could allow repair to occur. © 1993 Wiley‐Liss, Inc.
Effects of thymosin β4 and thymosin β10 on actin structures in living cells
Authors: Yu Fu‐Xin , Yin Helen L. , Morrison‐Bogorad Marcelle , Lin Sheng‐Cai 
Published: February 2005

AbstractThe β‐thymosins are a family of small proteins originally isolated from the thymus. Recently, two of the major mammalian isoforms, thymosin β4 (Tβ4) and thymosin β10 (Tβ10), are identified as significant actin monomer sequestering proteins which may be involved in regulating actin filament assembly. To study the cellular function of β‐thymosins, we have used isoform‐specific antibodies to determine their concentration and intracellular distribution, and examined the effects of inducing overexpression of Tβ4 and Tβ10 on actin filament structures. Immunofluorescence labeling of peritoneal macrophages showed that both β‐thymosins are uniformly distributed within the cytoplasm. cDNA‐mediated overexpression of β‐thymosins in CV1 fibroblasts induced extensive loss of phalloidinstained actin stress fibers. Stress fibers in the cell center were more susceptible than those at the periphery. There was a decrease in the number of focal adhesions, as evidenced by a decrease in discrete vinculin staining and an increase in diffuse vinculin fluorescence. The majority of the transfected cells had normal shape in spite of extensive loss of actin filaments. Occasionally, cells overexpressing β‐thymosin were observed to divide. In these cells, β‐thymosin was excluded from the midbody which contains an actin filament‐rich contractile ring. Our results indicate that Tβ4 and Tβ10 are functionally very similar and both are effective regulators of a large subset of actin filaments in living cells. © 1994 Wiley‐Liss, Inc.
Calmyonemin: A 23 kDa analogue of algal centrin occurring in contractile myonemes of Eudiplodinium maggii (ciliate)
Authors: David Christine , Viguès Bernard 
Published: February 2005

AbstractMyonemes are bundles of thin filaments (3‐6 nm in diameter) which mediate calcium‐induced contraction of the whole or only parts of the cell body in a number of protists. In Eudiplodinium maggii, a rumen ciliate which lacks a uniform ciliation of the cell body, myonemes converge toward the bases of apical ciliary zones that can be retracted under stress conditions, entailing immobilization of the cell. An mAB (A69) has been produced that identifies a calciumbinding protein by immunoblot, immunoprecipitation experiments and specifically labels the myonemes in immunoelectron microscopy. Solubility properties, apparent molecular weight (23 kDa) and isoelectric point (4.9) of the myonemal protein, are similar to the values reported for the calcium‐modulated contractile protein centrin. Western‐blot analysis indicates that the 23 kDa protein crossreacts antigenically with anti‐centrin antibodies. In addition, the 23 kDa protein displays calcium‐induced changes in both electrophoretic and chromatographic behaviour, and contains calcium‐binding domains that conform to the EF‐hand structure, as known for centrin. Based on these observations, we conclude that a calcium‐binding protein with major similarities to centrin occurs in the myonemes of E. maggii. We postulate that this protein plays an essential role in myonememediated retraction of the ciliature. © 1994 Wiley‐Liss, Inc.
ATP‐induced sliding of microtubules on tracks of 22S dynein molecules aligned with the same polarity
Authors: Mimori Y. , Miki‐Nomura T. 
Published: February 2005

AbstractChlamydomonas and Tetrahymena axonemal dyneins have previously been found to bind to porcine brain microtubules to produce a microtubule‐dynein complex. At appropriate microtubule:dynein concentration, microtubules in the complex became covered to saturation by dynein arms of the same polarity and at a spacing of 24 nm [Haimo et al., 1979; Haimo and Fenton, 1988; Haimo, 1989; Porter and Johnson, 1983a].In the present study, two different types of microtubule‐dynein complexes (α‐and β‐complexes) were prepared from Tetrahymena ciliary 22S dynein and porcine brain tubulin. The characteristics of the adenosine triphosphate (ATP)‐induced extrusion of microtubules from these complexes were analyzed, as a simple and direct in vitro assay for the ATP‐induced extrusion of single microtubules. The α‐complex prepared by adding dynein to microtubules showed an interrupted sliding movement, which would stop and start several times following the addition of ATP. In the β‐complex, prepared by adding dynein bound to DEAE‐tubulin to pre‐assembled microtubules, microtubules became covered with dynein molecules whose orientation and binding were uniform with respect to microtubule polarity. The microtubules in the β‐complex extruded at 12 μm/second following the addition of ATP. Dark‐field and electron microscopy indicated that the extruded microtubules had undergone sliding on a dynein‐track that had become detached from the complexes and had been absorbed onto the surface of the glass slide. At higher light intensity under a dark‐field microscope, the dynein‐track was seen to be composed of rows of dynein molecules arranged densely. The orientation of dynein molecules in rows appeared to be uniform considering the images of bound dynein in the β‐complex under electron microscope. The higher sliding velocity of the microtubules on these dynein‐tracks compared to that seen on slides coated at random with dynein [Vale and Toyoshima, 1988, 1989], may be due to more efficient force generation by this dense arrangement of dynein molecules with the same polarity on the tracks. © 1994 Wiley‐Liss, Inc.
Cellular infrared detector appears to be contained in the centrosome
Authors: Albrecht‐Buehler Guenter 
Published: February 2005

AbstractPrevious experiments have suggested that 3T3 cells were able to extend pseudopodia toward latex particles up to 60 μm away from the cell body if the particles were irradiated by an infrared beam in the range of 700–900 nm [Albrecht‐Buehler, 1991: J. Cell Biol. 114:493–502]. The present article reports that this response of cells to infrared light can be inhibited if the cell center is simultaneously irradiated with a beam of the same light. In marked contrast, the cells responded normally to the presence of infrared light scattering particles if the second beam irradiated other parts of the cell body. The results imply that the cellular mechanism of infrared detection is located at the cell center. The infrared sensing mechanism remains intact in enucleated cells and in cells which were incubated in monensin to vesiculate their Golgi apparatus and inhibit their Golgi functions. Accordingly, it is proposed that the centrosome which contains the centrioles is the only remaining candidate in the cell center for a cellular detection device for the direction of infrared signal sources. The results support an earlier suggestion that centrioles may be such detection devices [Albrecht‐Buehler, 1981: Cell Motil. Cytoskeleton 1:237–245]. © 1994 Wiley‐Liss, Inc.
Structural and geometrical constraints on the outer dynein arm in situ
Authors: Barkalow Kurt , Avolio Jock , Holwill Michael E. J. , Hamasaki Toshikazu , Satir Peter 
Published: February 2005

AbstractThis study considers the relationship between two structural forms of the 22S dynein arm of Tetrahymena thermophila: the bouquet and the compact arm. The compact arm differs from the bouquet and from other proposed forms (e.g., the “toadstool”) in that the globular domains are situated transversely across the interdoublet gap with one globular subunit, the head, proximal to the adjacent doublet microtubule. The other models place all three globular domains proximal to the neighboring doublet microtubule. When sliding of an isolated axoneme is induced, at least 57% of total attached arms on exposed doublets are in the compact form within dimensions of 24 × 24 × 12 nm, and only about 2% of the arms are bouquets. Toadstools are incompatible with the images seen. Bouquets are not found in regions of the doublet protected by a neighboring doublet. When axonemes with exposed doublets are treated with 0.5 M KCl for 30 min, the compct arms and the dynein heavy (H)‐chains disappear, while isolated bouquets and dynein H‐chains appear in the medium, suggesting that the compact arms give rise to the bouquets as they are solubilized. The bouquet is the predominant form of isolated 22S dynein molecules, which are found in two apparently enantiomorphic forms, within dimensions 45 × 39 × 13 nm; bouquets attached to doublets have dimensions similar to those of isolated bouquets. Computer modeling indicates that in an intact standard‐diameter axoneme, these dimensions are incompatible with the interdoublet volume available for an arm; the bouquet therefore represents an unfolded compact arm. A plausible sequence of changes can be modeled to illustrate the conversion of an attached compact arm to an attached and then free bouquet. The toadstool is probably an artifact that arises after unfolding. Consistent with the conformational difference, H‐chains of attached compact arms differ from those of isolated bouquets in their susceptibility to limited proteolysis. These results suggest that the compact arm, rather than the unfolded bouquet or the toadstool, is the functional form of the outer arm in the intact axoneme. © 1994 Wiley‐Liss, Inc.
Microtubule centers and the interphase microtubule cytoskeleton in amoebae of the cellular slime molds (mycetozoans) Acytostelium leptosomum and Protostelium mycophaga
Authors: Guhl Bruno , Roos Urs‐Peter 
Published: February 2005

AbstractWe investigated the microtubule (MT) cytoskeleton and microtubule centers (MTC) in undifferentiated amoebae by indirect immunofluorescence with six monoclonal antitubulin antibodies, and by transmission electron microscopy and immunogold ultracytochemistry. Interphase amoebae of both species contain a distinct cytoplasmic complex of MTs, which is more elaborate in Protostelium mycophaga. In Acytostelium leptosomum amoebae a single MTC is attached to each interphase nucleus at its pointed end, as in the other dictyostelid cellular slime molds Dictyostelium discoideum and Polysphondylium violaceum. Ultrastructurally, MTCs of A. leptosomum also resemble those of these two species: They consist of an electron‐opaque core shaped like a stout rod, which is embedded, together with nodules, in a fuzzy matrix. The nodules are the points of origin of the MTs. In most amoebae of P. mycophaga there are two MTCs on opposite sides of and close to the nucleus, but many amoebae also contain a variable number of MTCs that are remote from the nucleus. Nucleus‐associated and “remote” MTCs are structurally identical. They consist of a ring‐shaped core with inner and outer diameters of ca. 130 nm and 340 nm. A plug sits in the ring, and satellites are connected to the core by fine fibrils. The satellites are the points of origin of MTs. New MTCs are apparently formed during mitosis, the parent MTC probably serving as a template for the genesis of a new ring. The results support the notion that phylogenetically related organisms have similarly constructed MTCs and that these are dissimilar in less closely related organisms. © 1994 Wiley‐Liss, Inc.
pH‐Dependent solubility and assembly of microtubules in bovine brain extracts
Authors: Tiwari Suresh C. , Suprenant Kathy A. 
Published: February 2005

AbstractAlkaline pH favors the assembly of microtubules (MTs) in marine egg extracts [Suprenant and Marsh, 1987: J. Cell Sci. 184:167‐180; Suprenant, 1989: Exp. Cell Res. 184:167‐180; 1991: Cell Motil. Cytoskeleton 19:207‐220] and mammalian brain extracts [Tiwari and Suprenant, 1993: Anal. Biochem. 215:96‐103], even though the assembly of purified microtubule protein (MTP) from both of these sources is favored at slightly acidic pH. The present investigation examines whether alkaline pH has a direct or indirect effect on MT nucleation and growth in soluble brain extracts. Cell‐free extracts were prepared from bovine cerebral cortex, and a nucleated assembly assay was used to demonstrate that MT assembly in brain extracts is favored at slightly acidic pH. The increase in MT mass found at alkaline pH is due to an increase in the solubility of tubulin not an increase in the extent of assembly On average, 47.7 ± 11.3% of the total tubulin is soluble at pH 7.2, while only 30.9 ± 8.9% of the tubulin is soluble at pH 6.8. A model is proposed that indicates how microtubule proteins from both mammalian brain and marine eggs may be associated with pH‐dependent factors. © 1994 Wiley‐Liss, Inc.
Orientation, assembly, and stability of microtubule bundles induced by a fragment of tau protein
Authors: Brandt Roland , Lee Gloria 
Published: February 2005

AbstractThe neuronal microtubule‐associated protein tau has been implicated in the development of axonal morphology including the organization of microtubules into a uniformly oriented array of microtubules commonly referred to as “bundle.” Determination of the functional organization of tau has revealed that regions of tau protein which flank the microtubule‐binding domain affect the bundling of microtubules in vitro with a microtubule‐binding fragment of tau being most effective [Brandt and Lee, 1993: J. Biol. Chem. 268:3414‐3419]. In order to study the relation of microtubule bundles that form in vitro to those observed in the axon, we determined the orientation of individual microtubules in bundles and the effects of bundling on microtubule assembly and stability in cell‐free assembly reactions. Here we report that bundles induced by a microtubule‐binding fragment of tau contain randomly oriented microtubules as determined by using the difference in growth rates at microtubule plus and minus ends. We demonstrate that in vitro bundling increases microtubule growth (about 30%), stabilizes microtubules against dilution‐ and cold‐induced disassembly, and allows microtubule nucleation despite the absence of a tau region which has previously been shown to be required for tau‐dependent microtubule nucleation. We conclude that conditions that stabilize microtubules can lead to bundle formation and allow microtubule assembly by a mechanism different from that employed by microtubule‐associated proteins. The data also support the view that additional mechanisms besides the action of tau and tubulin exist in order to organize microtubules in the axon. © 1994 Wiley‐Liss, Inc.
Role of tropomyosin, α‐actinin, and actin binding protein 280 in stabilizing triton insoluble f‐actin in basal and chemotactic factor activated neutrophils
Authors: Watts Raymond G. , Howard Thomas H. 
Published: February 2005

AbstractF‐actin is a major component of the neutrophil (PMN) cytoskeleton. In basal PMNs, F‐actin exists in two structurally and functionally distinct pools: Triton insoluble F‐actin (TIF)–cold insensitive, not depolymerizable by dilution, and distributed in pseudopods and submembranous locations; and Triton soluble F‐actin (TSF)–unstable in cold, diffusely distributed, and gelsolin enriched. The element(s) conferring these unique properties to the Triton insoluble F‐actin pool are unknown, but logically include distinct actin regulatory proteins. To study the morphologic and functional determinants of the Triton insoluble F‐actin pool, the distribution and quantity of three candidate regulatory proteins, α‐actinin, tropomyosin (TM), and actin binding protein (ABP‐280), were compared in F‐actin (Triton insoluble and Triton soluble) and G‐actin pools isolated from basal and chemotactic factor activated human PMNs in suspension, using immunoblots and ionic extraction. F‐actin content was measured by NBDphallacidin binding and gel scans. The results show that: (1) α‐actinin, actin binding protein 280, and tropomyosin are localized to TIF and excluded from TSF; (2) TM, α‐actinin, and ABP 280 are required to stabilize fractions of Triton insoluble F‐actin in PMNs; and (3) chemotactic factor activation results in release of a fraction of TM from the Triton insoluble F‐actin pool in temporal association with F‐actin polymerization in the Triton insoluble F‐actin pool. Shifts in ABP 280 or α‐actinin do not occur. The results suggest that TM, α‐actinin, and ABP 280 provide structure to TIF and that TM release from TIF is involved in chemotactic factor induced actin polymerization in PMNs. © 1994 Wiley‐Liss, Inc.
Clustered acetylcholine receptors have two levels of organization in Xenopus muscle cells
Authors: Luther Paul W. , Samuelsson Steven J. , Bloch Robert J. , Pumplin David W. 
Published: February 2005

AbstractWe studied the organization of acetylcholine receptor (AChR) clusters by shearing cultured Xenopus muscle cells with a stream of buffer, and preparing rotary replicas of the exposed cytoplasmic surface of the sarcolemma. AChR clusters contained numerous particles that protruded from the sarcolemma and formed an irregular array composed of discrete aggregates. AChR were located within these particle aggregates, as shown by comparison of the replicas to labeling by fluorescent α‐bungarotoxin, and by immunogold cytochemistry with antibodies specific for the receptor. The aggregates were cross‐linked by a dense network of 7 nm filaments that replicated with the banded pattern characteristic of actin microfilaments. The organization of receptors into the small aggregates was independent of the organization of these aggregates into clusters, as alkaline extraction removed the microfilament network and disrupted the irregular array of particle aggregates, but did not disperse individual receptors from the aggregates. We conclude that two levels of interactions organize AChR clusters in Xenopus muscle cells: short‐range interactions that assemble individual AChR into small aggregates, and long‐range interactions, perhaps mediated by actin microfilaments, that anchor the aggregates into larger clusters. © 1994 Wiley‐Liss, Inc.
A conserved region in the tail domain of vimentin is involved in its assembly into intermediate filaments
Authors: Makarova Irina , Carpenter David , Khan Sohaib , Ip Wallace 
Published: February 2005

AbstractAlthough the head and rod domains of intermediate filament (IF) proteins are known to play significant roles in filament assembly, the role of the tail domain in this function is unclear and the available information supports contradictory conclusions. We examined this question by comparing transfection of the same cDNA constructs, encoding vimentins with modified tail domains, into cell lines that do and do not contain endogenous IF proteins. By this approach, we were able to distinguish between the ability of a mutant IF protein to initiate assembly de novo, from that of incorporating into existing filament networks. Vimentins with modifications at or near a highly conserved tripeptide, arg‐asp‐gly (RDG), of the tail domain incorporated into existing IF networks in vimentin‐expressing (vim+) cells, but were assembly‐incompetent in cells that did not express IF proteins (vim−). The failure of the RDG mutant vimentins to assemble into filament arrays in vim‐ cells was reversible by re‐introducing a wild‐type vimentin cDNA, whereupon both wild‐type and mutant vimentins coassembled into one and the same IF network. We conclude that the function of the tail domain of type III IF proteins, and possibly of keratins K8 and K18, in IF assembly is distinct from those of other domains; a region encompassing the RDG tripeptide appears to be important in the assembly process. © 1994 Wiley‐Liss, Inc.