bluuebunny/crossref_metadata_2025_split huggingface dataset:
Talin dynamics in living microinjected nonmuscle cells
Authors: Hock Rick S. , Sanger Jean M. , Sanger Joseph W. 
Published: February 2005

AbstractTo investigate the role of talin in the anchoring of actin‐containing stress fibers to the cell membrane of nonmuscle cells, a fluorescent analog of the adhesion plaque protein talin was developed, characterized, and microinjected into living cells. Purified chicken gizzard talin was covalently labeled with the fluorescent dye lissamine rhodamine B sulfonyl chloride. The fluorescently labeled protein was then chromatographed on Sephadex G‐25 and DEAE‐cellulose in order to remove free dye and denatured protein. The fluorescent talin was able to bind purified vinculin and was localized in adhesion plaques, membrane ruffles, microspikes, and polygonal networks in acetone‐permeabilized nonmuscle cells. In cells that were double‐stained with fluorescent talin and an affinity‐purified anti‐talin an‐tibody, a one‐to‐one correspondence of adhesion plaque staining was seen. Living epithelial cells (PtK2) were microinjected during interphase with fluorescent talin. Computer‐enhanced video microscopy was used to document adhesion plaque dynamics such as (1) changes in plaque shape, (2) alterations in plaque positions, and (3) the appearance, growth, and dissolution of plaques. In cells that were followed during mitosis, the adhesion plaques disappeared during cell rounding and then subsequently reappeared upon spreading of the two daughter cells. Treatment of microinjected cells with DMSO in order to disassemble stress fibers resulted in an altered localization of the fluorescent talin. Upon recovery of the cell from the drug, the talin was visualized in its characteristic submembraneous position. These results are the first to document the role and distribution of talin in dynamic processes occurring in living microinjected nonmuscle cells.
Splitting the ciliary axoneme: Implications for a “Switch‐Point” model of dynein arm activity in ciliary motion
Authors: Satir Peter , Matsuoka Tatsuomi 
Published: February 2005

AbstractIn the presence of specific inhibitors of beat, 20 μM VO43− or pCa 4, mussel gill lateral (L) cilia can be arrested in two positions—“hands down” or “hands up”—at opposite ends of the stroke cycle. Cilia move to these positions by doublet microtubule sliding. Axonemes of arrested cilia, still tethered to the cell, are intact after demembranation and protease treatment. When reactivated by 4 mM ATP with inhibitors present, about 40% split apart. Splits are not random but occur preferentially between different specific doublets in the two opposite arrest positions. Several different related patterns of splitting are observed; for every pattern in “hands down” axonemes, there is a corresponding complementary split pattern in “hands up” axonemes. In some split patterns two doublets remain firmly attached to the central pair; these also differ depending on axonemal position. Although some of the patterns seen may be artifactual or difficult to explain, the complementary splitting patterns are predictable with simple assumptions by a “switch point” hypothesis of ciliary activity where, during each recovery stroke, doublets 6–8 have active dynein arms, while during each effective stroke, arms on doublets 1–4 become active, and arms 6–8 are turned off. Because of a difference between the patterns seen and the predictions, the status of the arms on doublet 9 is unresolved. The patterns also suggest that a spokecentral sheath attachment cycle may correlate with switching of arm activity during the generation of an asymmetric beat.
Relationship between tektins and intermediate filament proteins: An immunological study
Authors: Steffen Walter , Linck Richard W. 
Published: February 2005

AbstractAffinity‐purified antibodies raised against three flagellar tektins (tektin A, B, and C) from each of two sea urchin species (Lytechinus pictus and Strongylocentrotus purpuratus) were used to study the immunological relationship between tektins and intermediate filament proteins. By immunofluorescence microscopy, several antitektins revealed a staining of intermediate filament‐like arrays in three vertebrate cell lines tested. Immunoelectron microscopy substantiated the cross reaction of antitektins with intermediate filaments. When the cells were treated with cytochalasin B, the arrangement of the filaments recognized by anti‐(Lp)‐tektin B was altered; the alteration observed is typical for keratin filaments. By immunoblot, it was found that anti‐(Lp)‐tektin B cross reacted with two isoforms or different proteins of ∼︁54 kD with pIs of 6.1 and 6.2 in human carcinoma epithelia (HeLa) cells and with two isoforms or different proteins of ∼︁55 kD with pIs of 6.1 and 6.3 in pig kidney epithelia (LLC‐PK1) cells. Furthermore, when antitektin antibodies were affinity purified with the 54 kD HeLa keratin, these keratin‐specific antibodies again restained the original tektins on immunoblots. From these observations, it can be concluded that tektins and keratins are to a certain extent immunologically related. To determine the degree of the immunological relationship, tektin filaments and purified intermediate filaments from HeLa cells were cleaved with α‐chymotrypsin and examined by quantitative immunoblot analysis. On immunoblots of digested tektins from L. pictus, anti‐(Lp)‐tektin B recognized several cleavage products in the range of 20 kD to 46 kD. However, when immunoblots of digested intermediate filaments from HeLa cells were probed, the cross reaction of anti‐(Lp)‐tektin B with HeLa keratins was eliminated by more than 98% within 2 min, suggesting that tektins have epitopes in common with the end domains of certain keratins.
Morphology of fibroblasts in collagen gels: A study using 400 keV electron microscopy and computer graphics
Authors: Heath Julian P. , Peachey Lee D. 
Published: February 2005

AbstractWe have used 400 kilovoit intermediate voltage electron microscopy (IVEM) to examine thick sections of fibroblasts cultured in collagen gels. In these 3D collagen lattices, the long, narrow pseudopodial extensions that extend out and make contact with the collagen matrix exhibit a complex topography not seen in the processes put out by cells moving on planar substrata. For this reason, sections 1 to 2 μm thick that enclose a whole cell process are more informative of the overall morphology of the interaction between cells and the collagen than are thin sections. To aid the discrimination of topography of cell processes in stereo views of micrographs, some cells were labeled with antibodies and protein A‐colloidal gold conjugates. The gold particles provided clear 3D reference points for computeraided reconstructions of membrane topography from tilt series of IVEM images. Our results confirm that cells that move through collagen lattices lack the wellspread morphology of their counterparts moving on glass. They are generally rather spindly with several long branching anterior pseudopodia. We found that the cell bodies and major pseudopodial processes were cylindrical, as one might expect of cells in a 3D environment, but at the leading edge of advancing pseudopodia there are small flat extensions similar to those seen in cells on glass. This similarity suggests that the lamellipodium is a basic type ofprotrusive structure used by fibroblasts during locomotion on all types of substratum. The flattened shape of lamellipodia may be part of the mechanism by which cells sense the orientation of fibrillar extracellular matrices during embryonic morphogenesis.
Isolation of a subpellicular microtubule protein from Trypanosoma Brucei that mediates crosslinking of microtubules
Authors: Balaban N. , Waithaka H. K. , Njogu A. R. , Goldman R. 
Published: February 2005

AbstractThe cell body of Trypanosomatidae is enclosed in densely packed, crosslinked, subpellicular microtubules closely underlying the plasma membrane. We isolated the subpellicular microtubules from bloodstream Trypanosoma brucei parasites by use of a zwitterion detergent. These cold stable structures were solubilized by a high ionic strength salt solution, and the soluble proteins that contained tubulin along with several other proteins were further fractionated by Mono S cation exchange column chromatography. Two distinct peaks were eluted containing one protein each, which had an apparent molecular weight of 52 kDa and 53 kDa. (Mr was determined by SDS‐gel electrophoresis.) Only the 52 kDa protein showed specific tubulin binding properties, which were demonstrated by exposure of nitrocellulose‐bound trypanosome proteins to brain tubulin. When this protein was added to brain tubulin in the presence of taxol and GTP, microtubule bundles were formed with regular crosslinks between the parallel closely packed microtubules. The crosslinks were about 7.2 nm apart (center to center). Under the same conditions, but with the 53 kDA protein or without trypanosome derived proteins, brain tubulin polymerized to single microtubles. It is thus suggested that the unique structural organization of the subpellicular microtubules is dictated by specific parasite proteins and is not an inherent property of the polymerizing tubulin. The in vitro reconstituted microtubule bundles are strikingly similar to the subpellicular microtubule network of the parasite.
Association of ribosomes with in vitro assembled microtubules
Authors: Suprenant Kathy A. , Tempero Libeth B. , Hammer Lorraine E. 
Published: February 2005

AbstractMicrotubules were purified from unfertilized eggs of the sea urchins Arbacia punctulata, Lytechinus pictus, Lytechinus variegatus, and Strongylocentrotus purpuratus. Numerous densely stained particles (24 × 26 nm) are associated with microtubules isolated from each of these sea urchins. The most striking aspect of this structure is an extended, slightly curved arm that appears to attach the particles to the microtubule. Morphologically similar particles are associated with microtubules of the isolated first cleavage mitotic apparatus. The particles are attached to the microtubules by ionic interactions and contain large amounts of extractable RNA. Based upon their size and density, RNA and protein composition, and sedimentation in sucrose gradients, the microtubule‐associated particles are identified as ribosomes.
Purification of anterogin, a protein factor necessary for the dispersion of carotenoid droplets in permeabilized xanthophores of goldfish
Authors: Zeng Zhao‐Chun , Taylor John D. , Tchen T. T. 
Published: February 2005

AbstractWe reported previously that the dispersion of carotenoid droplets in permeabilized xanthophores requires cAMP, ATP, and a cytosolic factor present in several secretory tissues as well as in xanthophores. We have now purified this factor from beef liver to apparent/near homogeneity. It appears to be a heterodimer with Mr ∼125,000. The purified factor has little or no ATPase activity, with or without the presence of actin. Nor does it stimulate the ATPase activity of carotenoid droplets. Its exact function in carotenoid droplet dispersion is thus unclear. Since dispersion of carotenoid droplets is an anterograde translocation, we propose the name anterogin for this protein. We also report that yeast cytosol has anterogin activity.
Light‐induced stop response in Chlamydomonas reinhardtii: Occurrence and adaptation phenomena
Authors: Hegemann Peter , Bruck Benjamin 
Published: February 2005

AbstractIn darkness Chlamydomonas cells swim forward with a helical motion of low amplitude. When cells are exposed to light conditions they are not adapted to, they perform direction changes or stop responses depending on how far the stimulant irradiance is shifted from the former adaptation level. Here we present the utility of a commercially available motion analysis system for the analysis of the Chlamydomonas stop response.Chlamydomonas cells stop in darkness only occasionally with a random temporal distribution but with a highly increased frequency after a flash or a step‐up light stimulation. The delay time, tD, defined as the minimal time difference between flash and a light‐induced stop, was below 50 ms. The reaction time, tR, defined as the time difference between flash and the maximal probability for a cell to stop was found to be 140 ms. During a stop the cells swim revers for some 300 ms with 20% of the forward swimming speed.To a given stimulation program cells adapt with a first‐order kinetic. In the case of a single step‐up or step‐down stimulation this adaptation consists of a single stop response followed by direction changes which decrease in frequency to a certain steady‐state level. To repetitive light pulses the cells respond with a gradual disappearance of step‐up stop responses and a concurrent appearance of step‐down responses.External calcium influences the stop response in a multifunctional way. For stop responses to occur 300 nM calcium are required. At increasing calcium concentrations the duration of a stop response is extended. Besides light, calcium regulates the time course of light‐adaption and the absolute adaptation level.A kinetic model for the description of adaptation is presented.
Nucleus‐basal body connector in Chlamydomonas: Evidence for a role in basal body segregation and against essential roles in mitosis or in determining cell polarity
Authors: Wright Robin L. , Adler Sally A. , Spanier Jonathan G. , Jarvik Jonathan W. 
Published: February 2005

AbstractIn the unicellular biflagellate green alga Chlamydomonas reinhardtii each basal body is linked to the nucleus by a fibrous nucleus‐basal body connector (NBBC) that contains the calcium‐binding protein centrin. (Wright et al.: Journal of Cell Biology 101:1903–1912.; Salisbury et al.: Journal of Cell Biology 107:635–642; Huang et al.: Journal of Cell Biology 107:121–131). In order to explore the cellular function of the NBBC we used antiserum directed against centrin to examine a number of mutants known to be defective for basal body assembly and/or localization. Of three variable flagella‐number mutants examined, one, vfl‐2, is dramatically defective with respect to the NBBC in that (1) the union between basal bodies and nucleus is very labile, (2) there is no detectible centrin in the NBBC region, and (3) total cellular centrin levels are reduced 75–80% relative to wild type. The existence of these defects in a mutant incapable of maintaining normal flagellar number supports the view that the NBBC plays an important role in determining proper basal body localization and/or segregation. In contrast to vfl‐2, the mutants vfl‐1, vfl‐3, uni‐1, and bald‐2 contain approximately normal levels of centrin and possess stable NBBCs. The observation of NBBCs in the mutant bald‐2, which lacks all but very rudimentary basal bodies, indicates that the assembly of the NBBC does not require fully formed basal bodies and that such assembly may not require basal bodies at all. Finally, the possibility that the NBBC is required for induction of gene expression following deflagellation was tested by examining vfl‐2 for such induction. Results indicate that the connector does not play a necessary role in the induction process.
Epidermal growth factor induces the accumulation of calpactin II on the cell surface during membrane ruffling
Authors: Campos‐Gonzalez Roberto , Kanemitsu Martha , Boynton Alton L. 
Published: February 2005

AbstractConfluent and proliferatively quiescent T51B rat liver epithelial cells provide a cellular model for the study of epidermal growth factor (EGF) effects in non‐neoplastic cells. Immunoreactive calpactin II, a well‐known substrate for EGF‐receptor kinase, was found predominantly in the cytosol, although a second im‐munoreactive pool was found in a Triton X‐100‐extractable membrane fraction. Stimulation with EGF resulted in a rapid and transient (2–;5 min) formation of ruffles at the cell surface and at the cell‐cell contacts. Both calpactin II and filamentous actin were found co‐localized at the membrane ruffles. Immunopre‐cipitations of membrane‐bound calpactin II from 32P‐labeled cells indicate a transient EGF‐dependent phosphorylation of calpactin II correlating with membrane ruffling. These results suggest a temporal (2–5 min) function for calpactin II at the plasma membrane during the EGF‐induced mitogenesis of T51B cells.
Purification and characterization of an 85 kDa talin‐binding fragment of vinculin
Authors: Groesch Mary E. , Otto Joann J. 
Published: February 2005

AbstractVinculin and talin are adhesion plaque proteins which have been shown to interact with each other in vitro. In order to begin to investigate where the talin‐binding domain is in vinculin, vinculin was digested with Staphylococcus aureus V8 protease to generate two major fragments of 85 and 30 kDa, and these fragments were purified. Nitrocellulose overlays with 125I‐talin and the 125I‐85 kDa vinculin fragment and sucrose density gradient centrifugation demonstrated that the talin‐binding domain was localized to the 85 kDa vinculin fragment. Quantification of 125I‐talin binding in the overlays showed that four times more talin bound to the 85 kDa fragment as compared to intact vinculin. Competitive immunoprecipitation experiments demonstrated that unlabeled 85 kDa fragment, was about three fold more effective at competing for 125I‐85 kDa binding to talin than was unlabeled vinculin. These results suggest that the 30 kDa fragment inhibits the vinculin‐talin interaction even though the talin‐binding domain is localized in the 85 kDa fragment.
Reorganization of cortical actin microfilaments and microtubules at preprophase and mitosis in wheat root‐tip cells: A double label immunofluorescence study
Authors: McCurdy David W. , Gunning Brian E. S. 
Published: February 2005

AbstractFollowing our recent description [McCurdy et al.: Protoplasma. 147:204–206, 1988] of arrays of transverse cortical microfilaments (MFs) in preprophase roottip cells of wheat (Triticum aestivum L. cv. Kite), we have performed double label immunofluorescence microscopy to correlate the formation of these arrays with the known rearrangement of cortical microtubules (MTs) that occurs during preprophase. At early preprophase, indicated by a broad (i.e., young) preprophase band (PPB) of MTs, actin MFs are transverse only in the central region of the cell cortex. By late preprophase, however, cells that possess a mature (i.e., narrow) PPB of MTs have arrays of transverse MFs that occupy the entire cortical surface of the cell. Thus, apart from the PPB zone, the transverse MFs in these arrays do not colocalize with transverse cortical MTs. Depolymerization of MTs using the herbicide oryzalin does not effect the arrays of cortical MFs; however, experiments using cytochalasin B in combination with oryzalin indicate that cellular MTs are necessary for the formation of the arrays of transverse cortical MFs. The arrays of cortical MFs disintegrate during prophase into short fragments of random, filamentous actin. This situation persists until the completion of cytokinesis. The absence of MFs during mitosis in densely‐cytoplasmic meristematic cells of wheat root tips indicates that filamentous actin may not have a universal function in plant cell division. The possible function of the arrays of cortical MFs in preprophase cells is discussed.
Effects of brain microtubule‐associated proteins on microtubule dynamics and the nucleating activity of centrosomes
Authors: Bré M. H. , Karsenti E. 
Published: February 2005

AbstractIn this paper, we report on the effect of brain microtubule‐associated proteins (MAPs) on the dynamic instability of microtubules as well as on the nucleation activity of purified centrosomes. Under our experimental conditions, tau and MAP2 have similar effects on microtubule nucleation and dynamic instability. Tau increases the apparent elongation rate of microtubules in proportion to its molar ratio to tubulin, and we present evidence indicating that this is due to a reduction of microtubule instability rather than to an increase of the on rate of tubulin subunits at the end of growing microtubules. Increasing the molar ratio of tau over tubulin leads also to an increase in the average number of microtubules nucleated percentrosome. This number remains constant with time. This suggests that the number of centrosome‐nucleated microtubules at steady state can be determined by factors that are not necessarily irreversibly bound to centrosomes but, rather, affect the dynamic properties of microtubules.
Partial characterization of a carotenoid droplet ATPase and its possible significance in carotenoid droplet dispersion in goldfish xanthophores
Authors: Wu Bei‐yue , Yu Fu‐xin , Lynch Thomas J. , Taylor John D. , Tchen T. T. 
Published: February 2005

AbstractThe dispersion of carotenoid droplets in permeabilized goldfish xanthophores is dependent on ATP, F‐actin, and cytosol. We report here that the motor (ATPase, translocator) resides with the permeabilized cell remnants and not in the cytosol. We also report that the carotenoid droplets have an ATPase that is not conventional myosin, dynein, or an ion pump. Its activity appears to correlate with the actin content of the carotenoid droplet preparation. A carotenoid droplet protein of Mr 72,000 (p72) is shown to be labeled by irradiation with 8‐azido‐ATP with concomitant loss of ATPase activity of the carotenoid droplets. We propose that this protein may be the ATPase responsible for carotenoid droplet dispersion.
A 70 kD microtubule‐binding protein from starfish eggs: Purification, characterization, and localization during meiosis and mitosis
Authors: Hosoya Natsumi , Hosoya Hiroshi , Mohri Toshiko , Mohri Hideo 
Published: February 2005

AbstractA microtubule‐binding protein was purified from eggs of the starfish, Asterias amurensis, through several steps of purification including the taxol‐dependent procedure [Vallee, 1982, J. Cell Biol. 92:435–442]. This protein consists of a single polypeptide chain having an apparent molecular mass of 70 kD determined by SDS‐PAGE. The 70 kD protein was identified as a unique microtubulebinding protein, judging from electrophoretic mobility, cleavage pattern by limited proteolysis, heat stability, and immunocrossreactivity. The 70 kD protein binds to brain and egg microtubules. It does not promote assembly of brain tubulin, but promotes that of egg tubulin in vitro in a concentration‐dependent manner.Using indirect immunofluorescence and immunoelectron microscopy with the anti‐70 kD protein antibody, we analyzed the cellular localization of the 70 kD protein in starfish oocytes and eggs during both meiotic maturation (meicsis) and first cleavage (mitosis). Immunofluorescence studies showed that the 70 kD protein localized on microtubule structures spread widely throughout the cytoplasm, the sperm aster, and the microtubules making up the mitotic apparatus through both meiosis and mitosis. The antibody, however, did not recognize sperm axonemes. These results were confirmed by immunoelectron microscopy. Using a colloidal gold technique, the 70 kD protein was localized along the microtubules in vivo.This 70 kD protein is the first microtubule‐binding protein that has been shown to localize along the microtubules in oocytes and eggs throughout meiosis and mitosis and to promote microtubule assembly. The 70 kD protein may be involved in the dynamic changes of microtubule structures occurring within oocytes and eggs.