bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cap100, a novel phosphatidylinositol 4,5‐ bisphosphate‐ regulated protein that caps actin filaments but does not nucleate actin assembly
Authors: Hofmann Andreas , Eichinger Ludwig , André Elisabeth , Rieger Daniela , Schleicher Michael 
Published: February 2005

AbstractThe fast and transient polymerization of actin in nonmuscle cells after stimulation with chemoattractants requires strong nucleation activities but also components that inhibit this process in resting cells. In this paper, we describe the purification and characterization of a new actin‐binding protein from Dictyostelium discoideum that exhibited strong F‐actin capping activity but did not nucleate actin assembly independently of the Ca2+ concentration. These properties led at physiological salt conditions to an inhibition of actin polymerization at a molar ratio of capping protein to actin below 1:1,000. The protein is a monomer, with a molecular mass of ∼ 100 kDa, and is present in growing and in developing amoebae. Based on its F‐actin capping function and its apparent molecular weight, we designated this monomeric protein cap 100. As shown by dilution‐induced depolymerization and by elongation assays, cap100 capped the barbed ends of actin filaments and did not sever F‐actin. In agreement with its capping activity, cap100 increased the critical concentration for actin polymerization. In excitation or emission scans of pyrene‐labeled G‐actin, the fluorescence was increased in the presence of cap100. This suggests a G‐actin binding activity for cap100. The capping activity could be completely inhibited by phosphatidylinositol 4,5‐bisphosphate (PIP2), and bound cap100 could be removed by PIP2. The inhibition by phosphatidylinositol and the Ca2+‐independent down‐regulation of spontaneous actin polymerization indicate that cap100 plays a role in balancing the G‐ and F‐actin pools of a resting cell. In the cytoplasm, the equilibrium would be shifted towards G‐actin, but, below the membrane where F‐actin is required, this activity would be inhibited by PIP2. © 1992 Wiley‐Liss, Inc.
Steric inhibition of cytoplasmic dynein and kinesin motility by MAP2
Authors: Lopez Luis A. , Sheetz Michael P. 
Published: February 2005

AbstractUsing several in vitro motility assays, we found that motility driven by the microtubule (MT) motors, kinesin and cytoplasmic dynein, could be inhibited by MAP2 but not by tau protein or the MT‐binding proteolytic fragment of MAP2.In MT gliding assays, even the presence of one MAP2 molecule per sixty‐nine tubulin dimers caused an inhibition of about 75% of MT motility at low concentrations of both motors. The percent inhibition of motility decreased with increasing concentration of either motor, suggesting that the inhibition was the result of competition for access to the MT surface. The decrease in the number of moving MTs with MAP2 was correlated with an increase in the frequency of release of moving MTs from the motor‐coated glass. In assays of in vitro vesicular organelle motility and formation of ER networks, the presence of MAP2 inhibited small vesicle movements and to a lesser extent ER network formation.To determine if competition for specific sites on the MT or coating of the MT surface inhibited motility, we used tau protein and the chymotryptic MT‐binding fragments of MAP2 to coat MTs. No inhibition was observed and there was even an increase in the number of attached and moving MTs in the gliding assay with tau‐coated MTs. Because MAP2, tau and the chymotryptic MT‐binding fragments of MAP2 bind to the same domain on tubulin, masking of the MT surface sites does not appear responsible for the inhibition of motility by MAP2. Rather, we suggest that the sidearm of MAP2 interfered with the interaction of motors with MTs and caused a dramatic increase in the rate of MT release. In vivo, MAP2 could play a major role in the generation of cellular polarity even at substoichiometric levels by inhibiting transport on microtubules in specific domains of the cytoplasm. © 1993 Wiley‐Liss, Inc.
Cytoskeletal sheets of mammalian eggs and embryos: A lattice‐like network of intermediate filaments
Authors: Capco David G. , Gallicano G. Ian , McGaughey Robert W. , Downing Kenneth H. , Larabell Carolyn A. 
Published: February 2005

AbstractMammalian eggs and embryos possess a major cytoskeletal network composed of large planar “sheets” distributed throughout the cytoplasm. Cytoskeletal sheets are found neither in mammalian somatic cells nor in eggs or embryos of non‐mammals. In this study, we have investigated the structural composition of the sheets in eggs and embryos of the golden Syrian hamster by (1) analysis of replicas from quick‐frozen, deep‐etched specimens, (2) analysis of thick, resinembedded specimens using an intermediate voltage electron microscope (IVEM), (3) laser diffraction of EM images, (4) differential extraction with detergents, and (5) immunocytochemistry. Our results indicate that each sheet is composed of two closely apposed arrays of 10‐nm filaments. Each filament within an array is held in register with its neighbor by lateral cross‐bridges and the two parallel arrays of filaments are interconnected by periodic cross‐bridges about 20 nm in length. Laser diffraction of negatives from IVEM images indicates that each array is composed of fibers that form a square lattice, and the two arrays are positioned in register by cross‐bridges forming a single sheet. This lattice forms the skeleton of the sheets which is covered with a tightly packed layer of particulate material. By incubation in media containing different ratios of mixed‐micelle detergents, it is possible to remove components sequentially from the sheets and to extract the particulate material. Immunocytochemical localization demonstrates that the sheets bind antibodies to keratin, and to a small extent actin, but do not bind antibodies to vimentin or tubulin. Examination of sheets within embryos at the time of embryonic compaction demonstrates that the sheets begin to fragment and disassemble in regions of blastomeres where desmosomes form, but undergo no structural alterations in interior and basal surfaces of the blastomeres. In regions of blastomere—blastomere contact the sheets fragment and associate with granules resembling keratohyalin granules found in keratinocytes. © 1993 Wiley‐Liss, Inc.
Actin‐binding protein is a component of bovine erythrocytes
Authors: Brown Kevin D. , Zinkowski Raymond P. , Hays Sean E. , Binder Lester I. 
Published: February 2005

AbstractActin‐binding protein (ABP) is a well‐characterized polypeptide capable of crosslinking filamentous actin. To date, this polypeptide has been shown to exist in a number of tissues and cultured cell lines. This report shows that by using a panel of three monoclonal antibodies for immunoblotting and immunofluorescence analysis, that ABP is present in bovine erythrocytes. Moreover, the data obtained suggest that this protein is a component of the erythrocyte membrane skeleton. Additionally, bovine erythrocyte ABP is shown to possess both an apparent molecular weight and an isoelectric point identical to that of bovine smooth muscle filamin, implying that these two polypeptides are identical. © 1993 Wiley‐Liss, Inc.
Microtubule‐associated motility in cytoplasmic extracts of sea urchin eggs
Authors: Gliksman Neal R. , Salmon E. D. 
Published: February 2005

AbstractWe have developed a method for producing sea urchin egg cytoplasmic extracts which support substantial microtubule‐associated motility, particularly minus end‐directed motility characteristic of cytoplasmic dynein. Particles translocated along microtubules and axonemes predominantly in the minus end direction; microtubules and axonemes glided across the coverslip surface only in the plus end direction (as expected for a minus‐end directed motor bound to the coverslip surface); and microtubules crosslinked into bundles in an antiparallel orientation. Velocities of particle and microtubule translocation were in the range of 0.5–1.8 μm/sec. Vanadate at 10 μM inhibited all gliding of the microtubules and axonemes, yet bidirectional particle transport persisted. Vanadate at concentrations of 25 μM and higher inhibited nearly all microtubule‐based motility in the preparation and produced parallel bundling of the microtubules. Motility was slowed but not stopped in the presence of 5 mM AMP‐PNP.Usually when a particle bound to a microtubule wall, it moved to the microtubule minus end. These particles often remained attached to the minus end. When a microtubule plus end in the shortening phase of dynamic instability reached a stationary particle on the microtubule, sometimes normal minus enddirected motility was activated, or at other times the particle remained attached to the shortening plus end. © 1993 Wiley‐Liss, Inc.
Microinjection of fluorescent brain tubulin reveals dynamic properties of cortical microtubules in living plant cells
Authors: Wasteneys Geoffrey O. , Gunning Brain E. S. , Hepler Peter K. 
Published: February 2005

AbstractFluorescent brain tubulin, injected into living cells of the green alga Nitella pseudoflabellata and the higher plant Tradescantia virginiana, incorporates into the cortical microtubules, allowing these structures to be observed. With confocal laser scanning microscopy, clear images of microtubules were recorded and changes in microtubule patterns documented. After injection, fluorescent lengths of microtubules appeared within a few minutes and their number and length increased rapidly to a “steady state” over the first 15 min. In many instances, fluorescent microtubules could still be detected several hours after injection. In the cells examined, microtubules are arranged as an array of separate units only occasionally displaying close association or accurate co‐alignment with neighboring microtubules. In what we perceive to be the steady state condition, some microtubules remain relatively static, while others undergo rapid changes in length or small translocations. We also document what appears to be bidirectional microtubule elongation during postdepolymerization assembly. © 1993 Wiley‐Liss, Inc.
Monensin‐sensitive cellular events modulate neurite extension on laminin: An example of higher‐order regulation of cell motility
Authors: Smalheiser Neil R. 
Published: February 2005

AbstractNG108‐15 cells extend “rapid‐onset” neurites vigorously within the first hour after plating in minimal serum‐free medium on Petri dishes coated with polylysine and laminin (1 ng/mm2). We recently reported that the initial rates of neurite formation and cell translocation are further accelerated in this system when nonspecific substratum attachment sites are partially blocked by polyglutamate, bovine serum albumin, or polyethylene glycol polymers [Smalheiser, N. R. (1991): Dev. Brain Res. 62:81–89]. When cells were plated in the presence of the monovalent cation ionophore monensin (1–5 μM) or hypertonic sucrose (50–100 mM), the initial rate of outgrowth on laminin/polylysine‐treated Petri dishes was not affected, yet the acceleration produced by polyglutamate was strongly inhibited. These data indicate that monensin‐sensitive intracellular events can regulate neurite extension on laminin indirectly, through modulating the effects exerted on cells by nonspecific substratum sites. Although the critical events affected by monensin remain to be identified, movements of laminin receptors (their clustering, internalization, and recycling) are likely targets for further study. © 1993 Wiley‐Liss, Inc.
FRAP analysis of the stability of the microtubule population along the neurites of chick sensory neurons
Authors: Edson Kathryn J. , Lim Soo‐Siang , Borisy Gary G. , Letourneau Paul C. 
Published: February 2005

AbstractIn order to study microtubule turnover in elongating neurites, chick embryo sensory neurons were microinjected with x‐rhodamine tubulin, and after 6–12 hours, short segments along chosen neurites were photobleached at multiple sites. Previous studies [Lim et al., 1989; 1990] indicated that recovery of fluorescence (FRAP) in neurites occurs by the dynamic turnover of stationary microtubules. In all cases, distal bleached zones recovered fluorescence faster than bleached zones more proximally located along the same neurites. Bleached zones at growth cones completely recovered in 30–40 minutes, while bleached zones located more proximally usually recovered in 50–120 minutes. In the most proximal regions of long neurites, recovery of fluorescence was often incomplete, indicating that a significant fraction of the microtubules in these regions were very stable. These studies indicate that there are differences in microtubule stability along the lenght of growing neurites. These differences may arise from the combined effects of (1) modifications that stabilize and lengthen microtubules in maturing neurites and (2) the dynamic instability of the distally oriented microtubule plus ends. © 1993 Wiley‐Liss, Inc.
Video microscopy of organelle inheritance and motility in budding yeast
Authors: Jones Heather D. , Schliwa Manfred , Drubin David G. 
Published: February 2005

AbstractBy adapting the time‐lapse video microscopy techniques that were developed for larger, more complex cells, to living Saccharomyces cerevisiae cells, intracellular organelle movements were observed. Differential interference contrast optics revealed an organelle transport process in cells treated with mating pheromone. Small particles were observed to travel distances of up to 6 μm at rates of 0.11–0.17 (and in one case 0.80) μm/sec. Overall, the frequency of these motile events was quite low compared to what is observed in cell types traditionally studied by video microscopy. The ability to discern clearly the vacuole and nucleus in budding yeast revealed the dynamics of these organelles and the fact that their movements are carefully orchestrated during the cell cycle. Two types of vacuolar dynamics were observed: (1) interconversion between one large organelle and numerous smaller organelles and (2) the formation of projections that extend from the mother cell's vacuole into the bub. When applied to the study of the many available cytoskeletal and cell cycle mutants, the application of video microscopy to the study of organelle movements in living yeast cells will provide a unique opportunity to determine the molecular mechanisms of intracellular motility and to elucidate the temporal controls over these processes. © 1993 Wiley‐Liss, Inc.
High level expression of nonacetylatable α‐tubulin in Chlamydomonas reinhardtii
Authors: Kozminski Keith G. , Diener Dennis R. , Rosenbaum Joel L. 
Published: February 2005

AbstractFollowing the discovery of acetylated α‐tubulin in the flagella of Chlamydomonas, many studies have documented the presence of acetylated α‐tubulin in a variety of evolutionarily divergent organisms. While this posttranslational modification may define an isoform with a unique function, the primary effect of α‐tubulin acetylation remains unknown. To study the function of α‐tubulin acetylation, we have transformed Chlamydomoas, a organism in which almost all of the flagellar tubulin ad a subset of the cytoplasmic microtubules are acetylated, with a α1‐tubulin gene whose product cannot be acetylated. Specifically, the codon for lysine 40, the lysine that is acetylated, has been replaced with the codons of nonacetylatable amino acids. To distinguish mutagenized α‐tubulin from that produced by the two endogenous α‐tubulin genes, mutant α‐tubulin was tagged with an epitope from influenza virus hemagglutinin. Utilizing the constitutive Chlamydomonas rubisco small subunit S2 promoter, we have obtained in selected clones high levels of nonacetylatable α‐tubulin expression approximating 50–70% of the total flagellar α‐tubulin. Immunofluorescence and immunoblot analysis of transformed cells indicated that nonacetylatable α‐tubulin could assemble, along with endogenous α‐tubulin, into both cytoplasmic and flagellar microtubules. However, no gross phenotypic effects were observed, suggesting that the effect of α‐tubulin acetylation is subtle. © 1993 Wiley‐Liss, Inc.
Ontogenesis of F‐actin in hair cells
Authors: Romand R. , Zine A.‐E. , Hafidi A. 
Published: February 2005

AbstractThis report describes the ontogenesis of cochlear stereocilia using scanning electron microscopy for analysis of cilia appearance, and fluorescence microscopy of phalloidin, a label for F‐actin, to determine the maturation of the cilia framework. Surface and frozen‐sectioned preparations of the otic capsule were obtained from several stages of rat pup development beginning at the 16th gestational day and at various stages until adulthood. In the earliest stage investigated, strong fluorescence labeling was visible on the apical part of Kölliker's organ, revealing a reticular outline of cell junctions. Hair cells started to differentiate at the 18th day of gestation from cells within the primordial receptor area. Phalloidin labeling revealed a sequential appearance of F‐actin as the hair cells differentiated from the cells within the Kölliker's organ. The differentiation of receptor cells occurred first with the appearance of a junctional complex between the hair cell and the surrounding cells. Then a cuticular plate appeared followed by the progressive emergence of stereocilia.The F‐actin labeling also revealed a progressive differentiation of receptors cells from the cochlear base to its apex. There was also an inner to outer hair cell developmental gradient of label. Inner hair cells developed stereocilia before outer hair cells. The third row of outer hair cells was the last to acquire stereocilia. The adult patern of stereocilia was reached around the 6th postnatal day.We conclude that the appearance of actin filaments in developing receptor cells and the emergence of stereocilia can be regarded as markers for correlating function and other structural differentiation. © 1993 Wiley‐Liss, Inc.
Motility of intracellular particles in rat fibroblasts is greatly enhanced by phorbol ester and by over‐expression of normal p21N‐ras
Authors: Alexandrova Antonina Y. , Dugina Vera B. , Paterson Hugh , Bershadsky Alexander D. , Vasiliev Jury M. 
Published: February 2005

AbstractParticle motility in cultured rat fibroblasts was studied using video‐enhanced differential interference contrast microscopy. The average velocity of large bright particles (apparent diameter about 0.5–0.7 μm) was measured in control cells and in cells treated with agents which affected targets related to signal transduction pathways. A Rat‐2‐derived fibroblast line transfected with a construct containing multiple copies of the N‐ras proto‐oncogene under the control of dexamethasonesensitive promoter was used as a main experimental model. Dexamethasone treatment was shown to induce high levels of N‐ras expression in these cells. This treatment greatly increased the average particle velocity. At the same time dexamethasone did not influence the particle motility in the non‐transfected parent cells and in the cells transfected with a construct which did not contain N‐ras. Phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C (PKC), also induced an approximate eightfold increase in the particle rate after several hours of incubation, while sphingosine, an inhibitor of PKC, prevented this activation. Sphingosine alone reduced the particle motility after a 20 min incubation. The particle movements were inhibited also by colcemid. These data show that the activation of N‐ras and PKC produced dramatic activation of microtubule‐dependent particle motility. A possible role of this activation in signal‐induced alterations of cell morphology is discussed. © 1993 Wiley‐Liss, Inc.
Microtubule behavior in PC12 neurites: Variable results obtained with photobleach technology
Authors: Keith Charles H. , Farmer Mark A. 
Published: February 2005

AbstractWe have examined the effects of various means of photobleaching on the recovery of fluorescene, movement, and morphology of the microtubules in the neurites of rhodamine‐tubulin‐injected PC12 cells. We find that, depending on power of and time of exposure to the bleaching beam, we can generate at least three different patterns of fluorescence recovery in regenerating PC12 neurites. If bleaching is performed with a relatively low‐power beam for an extended period, fluorescence in polymer recovers very little after 1 hours. Under these conditions, however, tubulin immunostaining is seen extending through the bleach zone, and microtubules are present through the bleached zone in thin section electron micrographs. If bleaching is performed with a high‐power laster, for 0.5–5 seconds, fluorescence recovery also is quite slow, but electron microscopic observations reveal that no microtubules extend through the bleached region of the neurite, and the uranyl acetate‐stained cytoplasm appears more electron lucent than in the unbleached neurite. Finally, if bleaching is performed by very brief exposure to a high‐intensity laser beam, resulting in an incomplete reduction of fluorescence intensity through the bleach zone, fluorescence recovery occurs within 20–30 minutes, and immunostained microtubules appear intact through the bleach zone; electron microscopy confirms that microtubules extended through the bleached zone of such neurites. In all three cases, movement of the bleach zone is observed in approximately half of the experimental neurites. These results indicate that highly variable microtubule behaviors can be obtained with photobleach technology, presumably due to different levels and pathways of photodamage generated by different bleach protocols. Nevertheless, it is clear that both turnover and movement of microtubules occur in FC12 neurites, and both are likely to be involved in neurite maintenance and growth. © 1993 Wiley‐Liss, Inc.
Contraction of reconstituted Dictyostelium cytoskeletons: An apparent role for higher order associations among myosin filaments
Authors: Aguado‐Velasco Carmen , Kuczmarski Edward R. 
Published: February 2005

AbstractA large number of cellular functions require assembly of actin and myosin and coordinated interactions between the resulting filaments. To better understand the structure and function of one such contractile assembly, we have begun fractionation and reconstitution studies of Dictyostelium cytoskeletons. Isolated cytoskeletons rapidly contracted when mixed with Mg‐ATP, and myosin II was essential for this since myosin‐depleted (stripped) cytoskeletons failed to contract. Dictyostelium, Acanthamoeba, or skeletal muscle myosins bound to stripped cytoskeletons with equal efficiency, and the Mg‐ATPase of all three myosins was stimulated by the cytoskeleton‐associated actin. Near neutral pH, however, only the homologous system reconstituted with Dictyostelium myosin contracted, despite the fact that under the same conditions all three myosins bound to myosin‐depleted (ghost) muscle myofibrils and restored contractility. Individual Dictyostelium myosin thick filaments have a strong tendency to aggregate and associate end‐to‐end, and this may be important for functional contraction of cytoskeletons. This suggestion is supported by the observation that under conditions where individual Acanthamoeba myosin filaments aggregated, reconstituted cytoskeletons contracted. None of the solution conditions tested caused rabbit muscle myosin filaments to aggregate or to contract cytoskeletons. Thus higher order associations among individual myosin filaments may be essential for some types of cell motility. © 1993 Wiley‐Liss, Inc.