bluuebunny/crossref_metadata_2025_split huggingface dataset:
Distribution of F‐actin elongation sites in lysed polymorphonuclear leukocytes parallels the distribution of endogenous F‐actin
Authors: Redmond Tim , Zigmond Sally H. 
Published: February 2005

AbstractWe compared, on lysed polymorphonuclear leukocytes (PMNs), the spatial distributions of sites that nucleate actin polymerization with the spatial distribution of endogenous F‐actin. Sites nucleating polymerization of exogenous actin were detected by incubating lysed cells with rhodamine‐labeled G‐actin under polymerizing conditions. Endogenous F‐actin was stabilized and stained by lysis of cells into fluorescein‐labeled (FITC) phalloidin. We found the distributions of rhodamine and fluorescein intensities in a given cell, resting or stimulated with chemoattractant, to be similar. Thus, after lysis the number of sites able to nucleate actin polymerization is proportional to the local F‐actin concentration.Quantitative fluorescence microscopic analysis also demonstrated that (1) if cells were stimulated with chemoattractant shortly before lysis, the total fluorescence per cell of both fluorophors went up; (2) if peptide was diluted shortly before lysis, the endogenous F‐actin in the lamellae was dramatically reduced, but nucleation sites persisted, giving a high rhodamine to fluorescein ratio; and (3) there was a small increase in the ratio of rhodamine (exogenously grown actin) to fluorescein (endogenous F‐actin) in a region near the lamellar/endoplasm border, centripetal to regions of the highest concentration of endogenous F‐actin.The rhodamine signal appeared to be due to in situ actin polymerization probably nucleated by existing free barbed ends, since (1) the rhodamine signal increased linearly with time with no detectable lag if the actin concentration was above that of the critical concentration of the barbed end; (2) the rhodamine signal was dramatically reduced if lysates were incubated with gelsolin–actin complex (which stably caps barbed ends), then washed before the rhodamine G‐actin was added; and (3) the number of nucleation sites at the time of lysis is similar to the number of the barbed ends of actin filaments determined by the kinetics of depolymerization [Cano et al., 1991].The fact that the distribution of exogenous actin polymerization paralleled the endogenous F‐actin suggests that the number of free barbed ends per F‐actin is roughly constant. If all filament ends were free, or if a constant fraction of the filaments ends were free, these data would suggest that the mean filament length is roughly constant throughout the cell. © 1993 Wiley‐Liss, Inc.
Computerized analysis of tumor cells flowing in a parallel plate chamber to determine their adhesion stabilization lag time
Authors: Patton John T. , Mentor David G. , Benson Douglas M. , Nicolson Garth L. , McIntire Larry V. 
Published: February 2005

AbstractThe importance of cell adhesion in a variety of physiological phenomena requires development of an understanding of the factors and molecular mechanisms underlying these behaviors. Cell adhesion is a multistep process involving primary receptor‐ligand interactions followed by secondary events that may lead to the formation of focal contacts. Due to the lack of well‐defined assays to study adhesion stabilization, little is known about this process, except that it may involve signaling events, receptor recruitment, and, as we have demonstrated, covalent peptide cross‐linking by cell membrane‐associated transglutaminase [Menter et al.: Cell Biophys. 18:123–143, 1992]. To study the stabilization process we have developed a dynamic assay employing a parallel plate flow chamber coupled with video microscopy and digital image processing. Our studies utilize wheat germ agglutinin‐selected human metastatic melanoma cell variants that exhibit differences in their experimental metastatic potential and expression of transglutaminase. Using this assay, quantifying cell‐substrate stabilization was found to be quick, reliable, reproducible, and useful in evaluating agents that block this process. © 1993 Wiley‐Liss, Inc.
Rat liver fat‐storing cell lines express sarcomeric myosin heavy chain mRNA and protein
Authors: Ogata Itsuro , Sáez Claudia G. , Greenwel Patricia , de Lourdes Ponce María , Geerts Albert , Leinwand Leslie A. , Rojkind Marcos 
Published: February 2005

AbstractFat‐storing cells (FSC, lipocytes, or Ito cells) of liver store vitamin A and are the main producers of extracellular matrix in normal and cirrhotic liver. During liver injury, FSC undergo an activation process characterized by a decrease in vitamin A storage and an increase in cell proliferation and extracellular matrix deposition. This activation process also occurs upon culturing FSC from normal liver. In contrast to most cells of nonmuscle origin, activated FSC express two cytoskeletal proteins normally found in muscle, desmin, and smooth muscle α‐actin. Based on their strategic perisinusoidal location, it has been hypothesized that FSC play a role in regulating blood flow. However, the nature of the contractile elements involved in this process remains to be determined. In this communication we demonstrate the presence of a sarcomeric myosin in proteins solubilized from liver biomatrix. In addition we demonstrate the expression of sarcomeric myosin heavy chain (MHC) mRNA and protein in two FSC clones derived from a CCl4‐cirrhotic rat liver (CFSC). Through cloning the cDNA corresponding to the MHC gene expressed in these cells we demonstrate that it encodes fast IId skeletal MHC and thus represents a marker normally seen in adult muscle. The unexpected expression of an adult stage skeletal muscle molecular motor in FSC from cirrhotic liver is consistent with the proposed specialized contractile capacity of these cells. © 1993 Wiley‐Liss, Inc.
Microtubule sliding in reduced‐amplitude bending waves of Ciona sperm flagella: Resolution of metachronous and synchronous sliding components of stable bending waves
Authors: Brokaw Charles J. 
Published: February 2005

AbstractMicrotubule sliding associated with the bending of reactivated flagella of demembranated spermatozoa of the tunicate, Ciona, has been analyzed using a descriptive model that permits quantitation of metachronous and synchronous components of sliding. Reduced‐amplitude bending waves, obtained by addition of increased salt (K acetate), lithium, or vanadate to the reactivation solutions, have been examined. Increased K acetate can decrease bend angle by as much as 70% with little change in frequency.In all cases, a decrease in the amplitude, or bend angle, of propagated bends is measured as a decrease in the metachronous component of sliding and is associated with a reduction in the growth of new bends after they begin to propagate during the second half‐cycle of bend development. At higher K acetate concentrations, bend growth during the second half‐cycle of bend development is very strongly reduced and may even become negative. A disparity between the rates of bend growth in the first and second half‐cycles of bend development corresponds to a large amount of synchronous sliding in the distal portion of the flagellum. When the synchronous sliding component is large, the sliding velocity in a propagating bend decreases to near‐0 values and may even reverse its direction as the bend propagates through the mid‐region of the flagellum. Since these large perturbations of sliding velocity do not interfere with regular propagation of bends with nearly constant bend angle, the bend propagation mechanism cannot operate by metachronous control of the velocity of sliding, and is unlikely to operate by local monitoring of either the amount or velocity of sliding. These observations therefore argue against models in which active sliding is regulated by shear or sliding velocity, and make curvature‐controlled models relatively more attractive.In many cases, a reduction in sliding during bend initiation (the first half‐cycle of development of new bends) also contributes to the decreased amplitude of propagated bends. These changes in bend initiation are similar in both full‐length flagella and in flagella shortened by breakage. The amount of sliding that occurs during bend initiation is relatively independent of the distribution of sliding between metachronous and synchronous components in the distal part of the flagellum. These observations therefore provide additional evidence that bend initiation and bend propagation are independent and separable processes. © 1993 Wiley‐Liss, Inc.
gCap39 is a nuclear and cytoplasmic protein
Authors: Onoda Koji , Yu Fu‐Xin , Yin Helen L. 
Published: February 2005

AbstractgCap39 is a newly identified member of the Ca2+‐ and polyphosphoinositidemodulated gelsolin family of actin binding proteins which is different from gelsolin in several important respects: it caps filament ends, it does not sever filaments, it binds reversibly to actin, it is phosphorylated in vivo, and it is also present in the nucleus. gCap39 and gelsolin coexist in a variety of cells. To better understand the roles of gCap39 and gelsolin, we have compared their relative amounts and intracellular distributions. We found that gCap39 is very abundant in macrophages (accounting for 0.6% of total macrophage proteins), and is present in 12‐fold molar excess to gelsolin. Both proteins are highly induced during differentiation of the promyelocytic leukemia cell line into macrophages. gCap39 is less abundant in fibroblasts (0.04% total proteins) and is present in equal molar ratio to gelsolin. The two proteins are colocalized in the cytoplasm, but gCap39 is also found in the nucleus while gelsolin is not. Nuclear gCap39 redistributes throughout the cytoplasm during mitosis and is excluded from regions containing chromosomes. Our results demonstrate that gCap39 is a nuclear and cytoplasmic protein which has unique as well as common functions compared with gelsolin. © 1993 Wiley‐Liss, Inc.
Unambiguous classification of microtubule‐ends in vitro: Dynamic properties of the plus‐ and minus‐ends
Authors: Kowalski Richard J. , Williams Robley C. 
Published: February 2005

AbstractTo understand the mechanism of dynamic instability of microtubule growth and shortening, one needs a means of reliably determining the polarity of the microtubules under investigation. Sea urchin sperm‐tail axonemal fragments nucleate the growth of both plus‐ended and minus‐ended microtubules, but their polarity is not apparent by video‐enhanced DIC microscopy. The polarity of a microtubule is usually assessed by observing differences between the rates and lengths of growth and shortening excursions of the two ends. In practice, though, a significant fraction of the population of microtubules displays characteristics intermediate between the average characteristics of either end, thereby escaping classification. Excluding these “intermediate” microtubules from the measured populations introduces bias into the understanding of microtubule dynamic instability. We circumvent this problem by making use of the plus‐end directed movement of the microtubule‐dependent molecular motor kinesin to determine the polarity of any given microtubule unambiguously. Carboxylated‐microspheres coated with kinesin, which are clearly visible by DIC microscopy, were used to determine the polarity of a microtubule. The dynamics were then observed. Kinesin was found to have no marked effect on dynamic instability. By this technique, we show that the distributions of properties that describe microtubule dynamic instability (rates and lengths of growth and shortening as well as frequencies of interconversion between these phases) of plus‐ends overlap to a significant extent with those of minus‐ends. It is this overlap that obscures the usual classification of the ends. Therefore, models describing microtubule dynamic instability need to incorporate the broad and overlapping range of properties of the two ends. © 1993 Wiley‐Liss, Inc.
Three‐dimensional dynamics of pseudopod formation and the regulation of turning during the motility cycle of Dictyostelium
Authors: Wessels Deborah , Vawter‐Hugart Holly , Murray John , Soll David R. 
Published: February 2005

AbstractEmploying a newly developed computer‐assisted system for visualizing and quantitating cell motility in three dimensions, we have examined the 3‐dimensional changes in cell shape and the dynamics of pseuodopod extension during translocation of Dictyostelium amoebae. Amoebae exhibit a 3‐dimensional behavior cycle with an average period of 1.5 min. The cycle includes a transient pseudopod extension phase in the x, y axis followed by a z‐axis expansion phase. Anterior pseudopod extension in the x, y axis is accompanied by a decrease in height, not by uropod retraction. The increase in height is accompanied by uropod retraction. In the pseudopod extension phase in the x, y axes, pseudopods form either anteriorly or laterally, and either on or above the substratum. Pseudopods which initially form on the substratum in almost all cases continue to expand as the anterior end of the cell. In the case of lateral pseuodopods, anteriorization leads to a turn. Approximately half of anterior pseudopod and two‐thirds of lateral pseudopods which initially form above the substratum are retracted. These results suggest that pseudopod‐substratum interaction plays a fundamental role in the regulation of directionality and turning in the translocation phase of the 3‐dimensional behavior cycle. © 1994 Wiley‐Liss, Inc.
Occurrence of fibers and their association with talin in the cleavage furrows of PtK2 cells
Authors: Sanger Jean M. , Dome Jeffrey S. , Hock Rick S. , Mittal Balraj , Sanger Joseph W. 
Published: February 2005

AbstractPtK2 cells of exceptionally large size were microinjected with fluorescently labeled probes for actin, myosin, filamin, and talin in order to follow the assembly of the contractile proteins into the cleavage furrows. Whereas in cells of normal size, there is usually a diffuse pattern of localization of proteins in the cleavage furrow, in these large, flat cells the labeled proteins localized in fibers in the cleavage furrow. Often, the fibers were striated in a pattern comparable to that measured in the stress fibers of the same cell type. The presence of talin in discrete plaques along fibers in the cleavage furrows of the large cells suggests a further similarity between cleavage furrow and stress fiber structure. The presence of filamin in the cleavage furrows also suggests the possibility of an overlapping mechanism in addition to that of a talin mediated mechanism for the attachment of actin filaments to the cell surfaces in the cleavage furrow. A model is presented that emphasizes the interrelationships between stress fibers, myofibrils, and cleavage furrows. © 1994 Wiley‐Liss, Inc.
Novel 130‐kDa rat liver myosin‐1 will translocate actin filaments
Authors: Williams Roger , Coluccio Lynne M. 
Published: February 2005

AbstractWe have recently purified and characterized from rat liver, polypeptides of 110‐kDa and 130‐kDa which possess several characteristics of myosin‐1 [Coluccio and Conaty: Cell Motil. Cytoskeleton 24:189‐199, 1993]. What roles these myosin‐1 molecules play in hepatocytes is not yet defined. One hypothesis is that they are involved in either intracellular transport or locomotion. As a first step in establishing their function, we have investigated whether these molecules are capable of supporting motility in vitro. Our results clearly demonstrate that the isolated 130‐kDa‐calmodulin complex will translocate filaments at a rate of 0.03‐0.05 μ/sec; motility is inhibited in free calcium ion concentrations above 0.1 μM. This inhibition is reversed with the addition of exogenous calmodulin. These results provide supporting evidence of a motile role for the 130‐kDa‐calmodulin complex in vivo. This is the first demonstration that in higher eukaryotes, myosin‐1 from a tissue other than intestine will support motility. Partial peptide sequence analysis indicates that the 130‐kDa polypeptide resembles the recently described myr 1 [Ruppert et al.: J. Cell Biol. 120:1393‐1403, 1993] or MM1α [Sherr et al.: J. Cell Biol. 1405‐1416, 1993] gene product. © 1994 Wiley‐Liss, Inc.
Role of MAPs and motors in the bundling and shimmering of native microtubules from insect ovarioles
Authors: Hunt Cherryl , Stebbings Howard 
Published: February 2005

AbstractBundles of native microtubules isolated from the ovarioles of hemipteran insects are seen to shimmer when observed using dark‐field microscopy. This novel form of microtubule motility becomes even more obvious when the isolated bundles are detergent‐extracted and reactivated. We have studied the nucleotide‐specificity and the drug‐sensitivity of microtubule shimmering in order to obtain information regarding the nature of the motor protein responsible, and to compare its properties with those of previously characterised microtubule motors. The involvement of structural MAPs in the shimmering and in maintenance of microtubule bundles in this system has also been investigated. © 1994 Wiley‐Liss, Inc.
Regulation of the Ascaris major sperm protein (MSP) cytoskeleton by intracellular pH
Authors: King Karen L. , Essig Juliane , Roberts Thomas M. , Moerland Timothy S. 
Published: February 2005

AbstractThe development and locomotion of the amoeboid sperm of the nematode, Ascaris suum, depend on precise control of the assembly of their unique major sperm protein (MSP) filament system. We used fluorescence ratio imaging of cells loaded with BCECF to show that intracellular pH (pHi) is involved in controlling MSP polymerization in vivo. Spermatogenesis is marked by a cycle of MSP assembly‐disassembly‐reassembly that coincides with changes in pHi. In spermatocytes, which contain MSP in paracrystalline fibrous bodies, pHi was 6.8, 0.6 units higher than in spermatids, which disassemble the fibrous bodies and contain no assemblies of MSP filaments. Activation of spermatids to complete development resulted in rapid increase in pHi to 6.4 and reappearance of filaments. Treatment of spermatocytes with weak acids caused the fibrous bodies to disassemble whereas incubation of spermatids in weak bases induced MSP assembly. The MSP filaments in spermatozoa are organized into fiber complexes that flow continuously rearward from the leading edge of the pseudopod. These cells established a pseudopodial pH gradient with pHi 0.15 units higher at the leading edge, where fiber complexes assemble, than at the base of the pseudopod, where disassembly occurs. Acidification of these cells caused the MSP cytoskeleton to disassemble and abolished the pH gradient. Acid removal resulted in reassembly of the cytoskeleton, re‐establishment of the pH gradient, and re‐initiation of motility. MSP assembly in sperm undergoing normal development and motility and in cells responding to chemical manipulation of pHi occurs preferentially at membranes. Thus, we propose that filament assembly in sperm is controlled by pH‐sensitive MSP‐membrane interaction. © 1994 Wiley‐Liss, Inc.
Functional analysis of a cardiac myosin rod in Dictyostelium discoideum
Authors: LeBlanc‐Straceski Janine M. , Fukui Yoshio , Sohn Regina L. , Spudich James A. , Leinwand Leslie A. 
Published: February 2005

AbstractManipulation of the single conventional myosin heavy chain (mhc) gene in Dictyostelium discoideum (Dd) has delineated an essential role for the filament‐forming, or light meromyosin (LMM) domain of the myosin molecule in cyto‐kinesis, development, and in the capping of cell surface receptors (see Spudich: Cell Regulation 1:1–11, 1989; Egelhoff et al.: Journal of Cell Biology, 112:677–688, 1991a). In order to assess the functional relationship between sarcomeric and cytoplasmic myosins, a chimeric gene encoding the Dd myosin head and subfragment 2 fused to rat β cardiac LMM was transfected into both wild‐type and Dd mhc null cells. Chimeric myosin was organized into dense cortical patches in the cytoplasm of both wild‐type and Dd mhc null cells. Although null cells expressing chimeric mhc at ∼10% of Dd mhc levels were unable to grow in shaking suspension or to complete development, chimeric myosin was able to rescue capping of cell surface receptors, to associate with filamentous actin, and to localize to the correct subcellular position during aggregation. Deletion of 29 amino acids in the rod corresponding to a previously defined filament assembly competent region eliminated the cortical patches and the posterior localization during chemotaxis. Taken together, these observations suggest that sarcomeric and cytoplasmic myosin rods are functionally interchangeable in several aspects of nonmuscle motility. © 1994 Wiley‐Liss, Inc.
Two‐step mechanism for actin polymerization in human erythroleukemia cells induced by phorbol ester
Authors: Niu M. Y. , Nachmias V. T. 
Published: February 2005

AbstractHuman erythroleukemia (HEL) cells grow in suspension, but after treatment with nM PMA the cells adhere and spread on glass or fibronectin [Jarvinen et al., 1987: Eur. J. Cell Biol. 44:238–246]. We observed an early (20–30 min) stage of spreading in which F‐actin was organized into peripheral arcs near the spreading margin and vinculin was localised to the cell's periphery at the ends of these arcs. By 1 h the cells were well spread with straight actin bundles many of which ended at more central sites terminating on patches containing vinculin and talin; thus the cells assemble typical stress fibers but do not appear to polarize. The cells also spread on RGD polymer. DiC8 (1,2‐dioctanoyl‐sn‐glycerol, C8:0, Sigma Chemical Co., St. Louis, MO) induced spreading but only if DAG kinase inhibitor and A‐23187 were also present; in their absence cells adhered but did not spread. Spreading was ∼85% inhibited by 100 nM staurosporine. PKC‐β was shown to be present in the cells by immunoblotting. In cells spread for 1 h with PMA, F‐actin increased to 180% of control levels as measured by RP binding and the actin sequestering complex of G‐actin‐thymosin β4 decreased significantly.To determine whether the F‐actin increase required adhesion, we inhibited cell attachment to the substratum by adding RGDS, by coating glass surfaces with hemoglobin, or by a combined treatment. Under these conditions PMA‐treated suspended cells still increased their F‐actin to 126–137% of controls, a significant increase over control levels. Staurosporine inhibited F‐actin increases under all the conditions studied.Permeabilized cell suspensions, incubated with rhodamine labelled G‐actin, incorporated the labelled actin along cell membranes at a low level. A few minutes preincubation with either diC8 plus DAG kinase inhibitor or with PMA strongly increased the incorporation. This increased incorporation was reduced to below control levels by either staurosporine (100 nM) or cytochalasin D (1 μM).We conclude that both suspended and spreading HEL cells can be stimulated to polymerize actin by a mechanism dependent on PKC or a PKC‐like molecule. In suspended cells, the polymerization occurs along the membrane. When cells spread, F‐actin increased to a significantly greater extent. This second step could involve additional polymerization, perhaps at the observed adhesion sites, decreased turnover of the actin bundles, or a combined effect of both mechanisms. © 1994 Wiley‐Liss, Inc.