bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microtubule associated protein MAP1A is an actin‐binding and crosslinking protein
Authors: Pedrotti Barbara , Colombo Roberto , Islam Khalid 
Published: February 2005

AbstractHigh molecular weight microtubule‐associated proteins MAP1A and MAP2 form thin projections from microtubule surfaces and have been implicated in crosslinking microtubules and other cytoskeletal components. We have purified native MAP1A from bovine brain and have studied its interaction with G‐ and F‐actin. Using a solid‐phase immunoassay we show that MAP1A binds in a dose‐dependent manner to both G‐actin and F‐actin. Addition of MAP1A to F‐actin causes gelation of F‐actin and SDS‐PAGE analysis shows that MAP1A co‐sediments with the gelled network, under conditions where F‐actin alone does not pellet. The low apparent viscosity of F‐actin is markedly increased in the presence of MAP1A, suggesting that MAP1A can crosslink F‐actin. Co‐incubation experiments indicate that MAP1A and MAP2 may bind to common or overlapping sites on the actin molecule. The widespread distribution of MAP1A and its interaction with microtubules, actin, and intermediate filaments suggests that it may constitute an important determinant of neuronal and non‐neuronal cellular morphology. © 1994 Wiley‐Liss, Inc.
22S axonemal dynein is preassembled and functional prior to being transported to and attached on the axonemes
Authors: Fok Agnes K. , Wang Hali , Katayama Akiko , Aihara Marilynn S. , Allen Richard D. 
Published: February 2005

AbstractIn an earlier study we reported the isolation of a cytoplasmic dynein from the cytosol of Paramecium multimicronucleatum. In this study we report the isolation and characterization of two cytosolic axonemal dyneins (22S and 12S) as well as a 19S cytoplasmic dynein from the cytosol of whole or deciliated cells using preformed bovine brain microtubules. These three dynein species were characterized according to mass, morphology, vanadate photocleavage patterns, CTPase/ATPase ratios, Km and Vmax values, temperature optima and reactivity with a mAb. For comparison, 22S and 12S axonemal dyneins (ADs) were also isolated and purified from the demembranated axonemes. The 22S and 12S soluble dyneins appear to be related to ciliary ADs in that the 22S soluble dynein is three‐headed while the 12S is a one‐headed dynein, as determined by negative staining. Ciliary ADs and their corresponding 22S and 12S soluble dyneins isolated from the cytosol also have similar Km and Vmax values as well as vanadate photocleavage patterns and temperature optima. A mAb raised against the soluble 22S dynein reacted with the 22S ciliary dyneins but not the 12S axonemal or the 19S cytoplasmic dynein. All isolated dyneins supported similar microtubule gliding rates but had different ionic requirements for the translocation buffer. These results suggest that: (i) the two soluble 22S and 12S dyneins are precursor molecules of the ciliary dyneins, (ii) the subunits of the outer arm dynein are already assembled in the cytosol as a three‐headed bouquet, and (iii) the 22S and 12S soluble dyneins are functional prior to being transported and attached to the axonemes of the cilia. © 1994 Wiley‐Liss, Inc.
Effect of protein kinase inhibitor H‐7 on the contractility, integrity, and membrane anchorage of the microfilament system
Authors: Volberg Tova , Geiger Benjamin , Citi Sandra , Bershadsky Alexander D. 
Published: February 2005

AbstractAddition of protein kinase inhibitor H‐7 leads to major changes in cell structure and dynamics. In previous studies [Citi, 1992: J. Cell Biol. 117:169–178] it was demonstrated that intercellular junctions in H‐7‐treated epithelial cells become calcium independent. To elucidate the mechanism responsible for this effect we have examined the morphology, dynamics, and cytoskeletal organization of various cultured cells following H‐7‐treatment. We show here that drug treated cells display an enhanced protrusive activity. Focal contact‐attached stress fibers and the associated myosin, vinculin, and talin deteriorated in such cells while actin, vinculin, and N‐cadherin associated with cell‐cell junctions were retained. Furthermore, we demonstrate that even before these cytoskeletal changes become apparent, H‐7 suppresses cellular contractility. Thus, short pretreatment with H‐7 leads to strong inhibition of the ATP‐induced contraction of saponin permeabilized cells. Comparison of H‐7 effects with those of other kinase inhibitors revealed that H‐7‐induced changes in cell shape, protrusional activity, and actin cytoskeleton structure are very similar to those induced by selective inhibitor of myosin light chain kinase, KT5926. Specific inhibitors of protein kinase C (Ro31‐8220 and GF109203X), on the other hand, did not induce similar alterations. These results suggest that the primary effect of H‐7 on cell morphology, motility, and junctional interactions may be attributed to the inhibition of actomyosin contraction. This effect may have multiple effects on cell behavior, including general reduction in cellular contractility, destruction of stress fibers, and an increase in lamellipodial activity. It is proposed that this reduction in tension also leads to the apparent stability of cell‐cell junctions in low‐calcium medium. © 1994 Wiley‐Liss, Inc.
Microtubules restrict plastid sedimentation in protonemata of the moss Ceratodon
Authors: Schwuchow Jochen , Sack Fred D. 
Published: February 2005

AbstractApical cells of protonemata of the moss Ceratodon purpureus are unusual among plant cells with sedimentation in that only some amyloplasts sediment and these do not fall completely to the bottom of vertical cells. To determine whether the cytoskeleton restricts plastid sedimentation, the effects of amiprophos‐methyl (APM) and cytochalasin D (CD) on plastid position were quantified. APM treatments of 30–60 min increased the plastid sedimentation that is normally seen along the length of untreated or control cells. Longer APM treatments often resulted in more dramatic plastid sedimentation, and in some cases almost all plastids sedimented to the lowermost point in the cell. In contrast, the microfilament inhibitor CD did not affect longitudinal plastid sedimentation compared to untreated cells, although it did disturb or eliminate plastid zonation in the tip. These data suggest that microtubules restrict the sedimentation of plastids along the length of the cell and that microtubules are load‐bearing for all the plastids in the apical cell. This demonstrates the importance of the cytoskeleton in maintaining organelle position and cell organization against the force of gravity. © 1994 Wiley‐Liss, Inc.
NI2+ inhibition induces asymmetry in axonemal functioning and bend initiation of bull sperm
Authors: Lindemann Charles B. , Walker Jay M. , Kanous Kathleen S. 
Published: February 2005

AbstractBull sperm extracted with 0.1% Triton X‐100 can be reactivated to full motility with 0.33 mM Mg‐ATP (sperm models). When motile sperm models are treated with 0.66 mM NiSO4, spontaneous motility is lost. During the transition to motility arrest, the beat becomes progressively more asymmetric, finally arresting at one extreme of the beat cycle. After spontaneous motility has been lost, the flagellum retains the ability to respond to mechanical stimulation. If a microprobe is used to bend the flagellum in the direction opposite to its own prevailing curvature and released, the recoil is rapid and overshoots the equilibrium position. When the same flagellum is manipulated in the opposite direction (into a tighter bend of the existing curve), the recoil is slower and does not exceed the initial bend. If a microprobe is used to carefully bend the whole flagellum into a curve, the flagellum will resume continuous beating, but only if the imposed bend is in the direction opposite the natural curvature. The reinstated beating activity (mechanical reactivation) is sustained as long as the flagellum is held by the microprobe. The rate of change of the shear angle in these mechanically reactivated, Ni2+ ‐inhibited sperm suggests an impaired rate of sliding on one side of the axoneme compared to similarly restrained control sperm. It appears that Ni2+ has a selective inhibitory effect on the dynein arms that bend the flagellum in one direction. Furthermore, the remaining functional arms activate only when the flagellum is bent in the direction opposing their own action. © 1995 Wiley‐Liss, Inc.
Phospholipid membrane‐associated brush border myosin‐I activity
Authors: Zot Henry G. 
Published: February 2005

AbstractBrush border myosin‐I (BBMI) is associated with the membrane of intestinal epithelial cells where it probably plays a structural role. BBMI also has been identified on Golgi‐derived vesicles in intestinal epithelial cells where it may translocate vesicles into the brush border. However, the mechanochemical activity of BBMI bound to a phospholipid membrane has not been described. This study reports that phospholipid membrane‐associated BBMI displays ATPase activity when bound to phospholipids, but does not move actin filaments when associated with a phospholipid bilayer. BBMI does not bind significantly to brush border membrane lipids, which contain about 16% phosphatidylserine (PS), in either a pelleting or planar membrane assay. Similarly, planar membranes containing 20% PS do not bind a significant amount of BBMI. Increasing the concentration of PS to 40% does result in the binding of BBMI to both vesicles and planar membranes. This binding is enhanced with increased Ca2+ concentrations. BBMI retains its ATPase activity when bound to phospholipid vesicles containing 40% PS. However, BBMI attached to a phospholipid bilayer surface does not move actin filaments, even though the amount of BBMI bound to the lipid surface, as reflected by the number of actin filaments associated with bilayer‐bound BBMI, is sufficient to observe motility in control experiments. When membrane fluidity is reduced by adding cholesterol to the membrane lipids containing 40% PS, BBMI still binds to the membrane, but again no actin filament motility is observed. The lack of binding by BBMI to brush border membrane lipids and the absence of membrane‐associated BBMI mechanical activity suggest that factors in addition to membrane lipids are necessary for membrane‐associated myosin‐I motility. © 1995 Wiley‐Liss, Inc.
Actin cytoskeleton of fibroblasts organizes surface proteoglycans that bind basic fibroblast growth factor and lipoprotein lipase
Authors: Fernáandez‐Borja Mar , Bellido David , Makiya Ricardo , David Guido , Olivecrona Gunilla , Reina Manuel , Vilaró Senén 
Published: February 2005

AbstractCell surface proteoglycans participate in molecular events that regulate cell adhesion, migration, and proliferation. To investigate the organization of these molecules at the cell surface, the distribution of two well‐known proteoglycan ligands has been studied. These ligands, lipoprotein lipase and basic fibroblast growth factor, showed a characteristic binding pattern consisting of highly organized parallel arrays that crossed the upper surface of human skin fibroblasts. The proteoglycan nature of the binding sites was evident from their susceptibility to heparinases, and from ligand displacement by heparin. Parallel localization of the ligands and actin, and treatment of the cells with cytochalasin, showed that the binding proteoglycans are organized by the actin cytoskeleton. The ligands induced a different behaviour of the binding sites on incubation of the cells at 37°C. Lipoprotein lipase produced a movement of the binding proteoglycans along the actin filaments towards the cell center. In contrast, after binding of basic fibroblast growth factor the binding proteoglycans remained spread over the cell surface and actin depolymerization was induced. Since an increasing number of ligands appear to depend on proteoglycans for their interactions with their high affinity receptors, distribution and movement of proteoglycans at the cell surface that is organized by the actin cytoskeleton could direct and enhance the encounters between the ligands and their specific receptors. © 1995 Wiley‐Liss, Inc.
Desmosome assembly and disassembly are regulated by reversible protein phosphorylation in cultured epithelial cells
Authors: Pasdar Manijeh , Li Zhi , Chan Honey 
Published: February 2005

AbstractDesmosomes are one component of the intercellular junctional complex in epithelia. In cultures of epithelial cells, desmosome assembly can be regulated by modulating the calcium concentrations of the growth media. At present, very little is known about the intracellular signal transduction mechanisms that regulate desmosome assembly and disassembly in response to changing extracellular calcium concentrations. We have used inhibitors of protein kinases and phosphatases in a combined biochemical and morphological approach to analyze the role of protein phosphorylation in the assembly and disassembly of desmosomes in Madin‐Darby canine kidney epithelial cells. Our results suggest that desmosomal proteins (desmoplakins I/II and desmoglein 1) are primarily phosphorylared on serine residues. Electron microscopic analyses of desmosome assembly upon induction of cell‐cell contact, in the presence of protein kinase inhibitor, H‐7, revealed an apparently normal assembly of desmosomes. However, complete disassembly of desmosomes was inhibited by H‐7 upon removal of extracellular calcium. Under these conditions, although desmosomes split, desmosomal plaques and their associated cytokeratin filaments can not be internalized. In contrast, treatment of the cultures with okadaic acid (OA), an inhibitor of protein phosphatases, inhibited desmosome assembly but had no effect on disassembly. In addition, the inhibitory effect of okadaic acid on desmosome assembly was specific to this junction since we observed apparently normal tight junction and adherens junction in okadaic acid‐treated cultures. These results suggest that via reversible protein phosphorylation involving both protein kinase and protein phosphatases. © 1995 Wiley‐Liss, Inc.
Quantitation of cap Z in conventional actin preparations and methods for further purification of actin
Authors: Casella James F. , Barron‐Casella Emily A. , Torres Michelle A. 
Published: February 2005

AbstractGel‐filtration is commonly used to remove contaminants from conventional actin prepared by the method of Spudich and Watt. It has been shown that this procedure removes the majority of a factor that reduces the low‐shear viscosity of actin. We have previously reported that this factor is Cap Z, a barbed end capping protein. We now establish that, even after gel‐filtration, enough Cap Z can be present in conventionally prepared actin to affect events occurring at the barbed ends of actin filaments. We also demonstrate that the concentration of Cap Z can be reduced to more than a log below the KD for binding of Cap Z to actin by either (1) immunoabsorbtion of conventionally prepared actin with anti‐Cap Z antibodies, or (2) an additional cycle of polymerization/depolymerization followed by repeat gel‐filtration. © 1995 Wiley‐Liss, Inc.
Purification of microtubule associated protein MAP1B from bovine brain: MAP1B binds to microtubules but not to microfilaments
Authors: Pedrotti Barbara , Islam Khalid 
Published: February 2005

AbstractA simple procedure for the purification of MAP1B from bovine brain is described. The procedure requires two ion‐exchange chromatographic steps and results in >95% pure MAP1B with a typical recovery of about 25–30 mg/kg of brain tissue. SDS‐PAGE analysis of the purified protein shows that it is composed of a high molecular mass (330kDa) heavy chain and two low molecular mass (32kDa and 18kDa) associated light chains. The estimated stoichiometry of heavy chain:light chain is 1:2 and 1:0.2 mole/mole protein for the 32kDa and 18kDa light chains respectively. Western blotting, using monospecific monoclonal antibodies, shows that only the heavy chain is recognised by the anti‐MAP1B antibody and is not immunostained by either the MAP1A or MAP2 monoclonal antibodies. Purified MAP1B binds efficiently to both unpolymerised tubulin and polymerised tubulin and co‐sediments with taxol‐stabilised microtubules. Co‐incubation experiments show that MAP2 can compete with MAP1B binding to microtubules, indicating common or overlapping sites. However, MAP1B binds to neither G‐actin nor F‐actin nor co‐sediments with F‐actin, suggesting that it is not an actin‐binding protein.
Expression of kinesin heavy chain isoforms in retinal pigment epithelial cells
Authors: King‐Smith Christina , Bost‐Usinger Laurie , Burnside Beth 
Published: February 2005

AbstractTo examine the possible role of kinesin in pigment granule migration in the retinal pigment epithelium (RPE) of teleosts, we investigated the expression and distribution of kinesin heavy chain (KHC) in RPE. Blots of fish RPE lysates probed with two well‐characterized antibodies to KHC (H2 and HD) displayed a prominent band at 120 kD. A third KHC antibody (SUK4) recognized a band at 118 suggesting the presence of two KHC isoforms in teleost RPE. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) of mRNA from RPE using primers homologous to conserved regions of the KHC motor domain resulted in the homologous to conserved regions of the KHC motor domain resulted in the identification of two putative KHC genes (FKIF1 and FKIF5) based on partial amino acid sequences. Previous studies had demonstrated a requirement for microtubules in pigment granule aggregation in RPE. In addition, the reported microtubule polarity orientation in RPE apical projections is consistent with a role for kinesin in pigment granule aggregation. Immunofluorescent localization of KHC in isolated RPE cells using H2 revealed a mottled distribution over the entire cell body, with no detectable selective association with pigment granules, even in cells fixed while aggregating pigment granules. Microinjected KHC antibodies had no effect on pigment granule aggregation or dispersion, although each of the three antibodies has been shown to block kinesin function in other systems. Thus we found no evidence for KHC function in RPE pigment granule aggregation. However, the two KHC isoforms may participate in other microtubule‐dependent processes in RPE.
Strikingly different propulsive forces generated by different dynein‐deficient mutants in viscous media
Authors: Minoura Itsushi , Kamiya Ritsu 
Published: February 2005

AbstractThe propulsive force generated by Chlamydomonas mutants deficient in flagellar dynein was estimated from their swimming velocities in viscous media. The force produced by wild‐type cell increased by 30–40% when viscosity was raised from 0.9 to 2 cP but decreased as viscosity was further raised above 6 cP. The biphasic dependence of force generation on viscosity was also observed in the mutant idal, which lacks the II component of the inner‐arm dynein. The mutant ida4, which lacks the inner‐arm 12 component, was extremely susceptible to viscosity and stopped swimming at 6 cP, at which other mutants could swim. In contrast, odal, which lacks the entire dynein outer arm, produced a fairly constant force of about one‐third of the wild‐type value, over a viscosity range of 0.9–11 cP. In demembranated and reactivated cell models of the wild type, the propulsive force decreased monotonically as viscosity increased. Thus the increase in force generation at about 2 cP observed in live cells may be caused by some unknown mechanism that is lost in cell models. The cell models of odal, in contrast, did not show a marked change in force generation with the change in viscosity. These results indicate that the force generation by the outer‐arm dynein greatly depends on viscosity or the velocity of movement, whereas the complete set of inner‐arm dynein present in the odal axoneme produces a fairly constant force at different viscosities. These different properties of inner and outer dynein arms should be important in the mechanism that produces flagellar beating.
Novel mode of hyper‐oscillation in the paralyzed axoneme of a chlamydomonas mutant lacking the central‐pair microtubules
Authors: Yagi Toshiki , Kamiya Ritsu 
Published: February 2005

AbstractThe flageliar axoneme of the mutant pf18 lacking the central pair does not beat, but undergoes a nanometer‐scale, high‐frequency oscillation (hyper‐oscillation) in the presence of ATP [Yagi et al., 1994: Cell Motil, Cytoskeleton 29:177–185]. The present study demonstrates that the amplitude of the hyper‐oscillation increases significantly in the simultaneous presence of ATP and ADP. In addition, the hyper‐oscillation under these conditions sometimes takes on an exceptionally simple asymmetric pattern, in which the maximal shearing velocity exceeds 50 μm/sec, much higher than the maximal velocity of ordinary dynein‐microtubule sliding. The asymmetric oscillation thus appears to be at least partly driven by an internal elastic force. Its amplitude suggests that the axoneme has an elastic component that can be stretched by as long as 0.1 μm. Analyses of the asymmetric pattern further suggests that the axonemal dyneins have a tendency to attach to and detach from the doublets cooperatively and that the mechanochemical cycle of dynein has an inherent refractory period of about 2 msec, during which dynein cannot interact with microtubules.
Traction forces in locomoting cells
Authors: Oliver Tim , Jacobson Ken , Dembo Micah 
Published: February 2005

AbstractA means of determining quantitative maps of the tractions exerted by locomoting cells on a substratum has been developed. This method is similar to the Harris silicone substratum assay [Harris et al., 1980: Science 208:177–179], but uses an improved non‐wrinkling film that deforms more predictably in response to traction forces. The method also utilizes a mathematical analysis of rubber deformation to produce the final map of the distribution of tractions. The resulting maps consistently showed that fish keratocytes exert a steady‐state “pinching” on the substratum, perpendicular to the cell's direction of locomotion. No significant rearward tractions were detected at or near the front edge of the cell. Likewise, no significant forward tractions associated with peeling of adhesions were found at the back of the cell. A second assay uses deflection of a lightly attached glass microneedle to measure the total force exerted by locomoting cells. Forces of approximately 4.5 × 10−3 dyn were required to “stall” locomoting keratocytes. The implications of these findings for cell movement are discussed.