bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assembly dynamics of actin in adherent human neutrophils
Authors: Wang Jia‐Sheng , Pavlotsky Nelli , Tauber Alfred I. , Zaner Ken S. 
Published: February 2005

AbstractWe have extended our previous studies of adherent neutrophils and compared actin depolymerization and intracellular calcium changes induced by adherence to laminin and fibronectin. In order to accurately assess cellular actin changes, F‐actin depolymerization in the cell lysates must be inhibited. We found that phalloidin or 3.7% formaldehyde treatment effectively inhibited the depolymerization of F‐actin fragments following cell lysis. Formaldehyde and phalloidin treatment reduced G‐actin levels 75–80% in suspended cells, 35–73% in cells adherent for 1 min, and about 50% for cells adherent for 3 min. When the actin was fixed, there were highly significant differences in G‐actin levels between the suspended and adherent cells as compared with unfixed cells. Adhesion to both laminin and fibronectin initiated a rapid rise in G‐actin with a corresponding decrease in F‐actin. However, the changes were more pronounced in cells adherent to laminin. The peak of depolymerization occurred by 1 min and, thereafter, G‐actin decreased and F‐actin increased reaching a steady state at 5 min. Adhesion to both laminin‐ and fibronectin‐coated surfaces was accompanied by an increase of [Ca2+]i with a peak at 3 min, followed by a decrease from 3–5 min and a steady state attained between 5 and 10 min. The rise of [Ca2+]i in laminin‐adherent cells was about twice that in fibronectin‐adherent cells at 3 min (P < 0.02). Pertussis toxin, H‐7, and staurosporin treatments did not alter the dynamic changes of actin in adherent cells, suggesting that these metabolic events are transduced by a G‐protein and Protein Kinase C independent mechanism. The results support the hypothesis that a transient mobilization of F‐actin to a monomeric pool, which then serves as a source for further repolymerization, is induced by adherence of neutrophils to extracellular matrix proteins. © 1993 Wiley‐Liss, Inc.
Dithiothreitol prevents membrane fusion but not centrosome or microtubule organization during the first cell cycles in sea urchins
Authors: Schatten Heide 
Published: February 2005

AbstractDithiothreitol (DTT), a disulfide reducing agent, inhibits the fusion of male and female pronuclei within the activated cytoplasm of sea urchin eggs. The migrations of the pronuclei are not affected by DTT, indicating that microtubule function is not impaired. Centrosomal antigens are detected in the sperm aster and in all subsequent microtubule‐based configurations. Nuclear membranes never fuse and the chromatin of male and female pronuclei never mix in the DTT‐treated cells. During prophase, when nuclear envelopes break down to undergo mitosis, both sets of chromosomes undergo condensation cycles independent from each other. Both pronuclei initially stain for centrosomal material and surrounding microtubules. With time, the female's centrosomal material as well as the microtubules disappear while the male forms a bipolar spindle. Interestingly, one pole of the paternal mitotic apparatus communicates with the separate maternal chromatin, forming a half spindle which moves the egg‐derived chromatin towards its pole. At the time for cell division, the individual karyomeres are not able to fuse their nuclear membranes to reconstitute the blastomere nuclei. When DTT is applied at prometaphase of the first cell cycle, the chromosome cycle continues until next metaphase. Centrosomes also continue their cycle and undergo somewhat atypical splitting during the time for second telophase. Division furrows are initiated but aborted. These results support the hypothesis that disulfide groups are required for membrane fusion of the pronuclei, for membrane fusion of the karyomeres, and for the completion of the division furrow to achieve successful cell division. © 1994 Wiley‐Liss, Inc.
Microtubules with altered assembly kinetics have a decreased rate of kinesin‐based transport
Authors: Redenbach Darlene M. , Richburg John H. , Boekelheide Kim 
Published: February 2005

AbstractMicrotubules treated with the γ‐diketone 2,5‐hexanedione (2,5‐HD) have altered assembly behavior characterized by precocious nucleation and rapid elongation. By measuring the rate of microtubule transport, we have examined the potential functional significance of this 2,5‐HD‐induced microtubule modification. 2,5‐HD‐treated microtubules were transported at only 70% of the rate of control microtubules in a simple kinesin‐based motility assay on glass coverslips using video and computer enhanced differential interference contrast microscopy. Since 2,5‐HD is capable of forming both pyrrole adducts and crosslinks with tubulin, the contributions of pyrrole formation and crosslinking to slowed microtubule transport were determined. 3‐Acetyl‐2,5‐hexanedione (AcHD), a pyrrole forming, non‐crosslinking congener of 2,5‐HD which does not alter microtubule assembly, did not produce slowed microtubule transport as occurs with 2,5‐HD. However, glutaraldehyde, a pyrrole‐independent crosslinking agent which alters microtubule assembly in the same way as 2,5‐HD, slowed microtubule transport. These results indicate that a 2,5‐HD‐induced microtubule modification, possibly a crosslink‐related conformational change, produces both an alteration in the kinetics of assembly and an alteration in the microtubule‐motor interaction. © 1994 Wiley‐Liss, Inc.
Control of flagellar bending: A new agenda based on dynein diversity
Authors: Brokaw Charles J. 
Published: February 2005

AbstractObservations that were interpreted to provide evidence for equivalent functions of all axonemal dyneins should be reinterpreted, and models based on this assumption should be abandoned. In the future, attempts to understand the mechanisms for flagellar bending, oscillation, and bend propagation should start from the assumption that each type of axonemal dynein may have a specific function. At least three distinct functions can now be identified: bend initiation, maintenance of the angle of propagating bends, and generation of power to overcome viscous resistances. Only the last of these three functions is an outer arm dynein function. © 1994 Wiley‐Liss, Inc.
Cytoplasmic dynein binds to phospholipid vesicles
Authors: Lacey Merri Lynn , Haimo Leah T. 
Published: February 2005

AbstractCytoplasmic dynein is the putative motor protein for retrograde organelle transport along microtubules in cells and, thus, must be capable of binding to organelle membranes. Such an attachment may occur via receptor proteins or through a direct interaction of dynein with the membrane phospholipids. We show here that cytoplasmic dynein‐synaptic membrane binding does not require a receptor protein and that this binding is mediated by an electrostatic interaction with acidic phospholipids. The properties of cytoplasmic dynein binding to NaOH‐extracted synaptic membranes are not significantly affected when those membranes are treated with trypsin to digest endogenous integral membrane proteins. Moreover, purified cytoplasmic dynein is capable of binding to liposomes composed of pure phospholipids. Dynein binds to liposomes with a profile remarkably similar to that of dynein binding to native membranes. Dynein‐liposome binding is dependent upon the presence of acidic phospholipids and is disrupted by NaCl. Thus, these studies suggest that electrostatic interactions can effect dynein‐membrane binding. © 1994 Wiley‐Liss, Inc.
Metachronal activity of cultured mucociliary epithelium under normal and stimulated conditions
Authors: Gheber Larisa , Priel Zvi 
Published: February 2005

AbstractIn the present work we measured in real time the metachronism and degree of correlation between beating cilia from cultured mucociliary epithelium. The method is based on simultaneous measurement of ciliary beat frequency, phase shifts, and correlation factors in two directions: parallel and perpendicular to the effective stroke direction (ESD). From the phase shifts the lengths of wave components, and consequently the metachronal wavelength and direction, were evaluated.On active ciliary areas of cultured frog esophagus under normal conditions, a relatively high degree of correlation is observed, but cilia are more correlated in direction parallel to ESD which is also the direction of the mucus propulsion. The length of the wave component parallel to ESD is more than twice as large as that of the perpendicular component. The metachronal wavelength was found to be in the range of 5–9 μm, and the direction of the wave propagation was in the range of 90°–125° clockwise to the ESD.When ciliary beat frequency was rapidly increased by extracellular ATP or acetylcholine, only minor effects were observed on the degree of correlation between beating cilia. The length of the wave component parallel to ESD showed the most dramatic effect increasing up to tenfold. The perpendicular to ESD component was not affected by the stimulation. Consequently, the metachronism became more laeoplectic with the angle between the ESD and the wave directions decreasing by 10°–30°, and the metachronal wavelength remained unaltered. © 1994 Wiley‐Liss, Inc.
A model of flagellar and ciliary functioning which uses the forces transverse to the axoneme as the regulator of dynein activation
Authors: Lindemann Charles B. 
Published: February 2005

AbstractCiliary and flagellar motion is driven by the dynein‐tubulin interaction between adjacent doublets of the axoneme, and the resulting sliding displacements are converted into axonemal bends that are propagated. When the axoneme is bent in the normal beating plane, force develops across the axoneme in the plane of the bend. This transverse force (t‐force) has maximal effect on the interdoublet spacing of outer doublets 2–4 on one side of the axoneme and doublets 7–9 on the opposite side. Episodes of sliding originates as the t‐force brings these doublets into closer proximity (allowing dynein bridges to form) and are terminated when these doublets are separated from each other by the t‐force. A second factor, the adhesive force of the dynein‐tubulin attachments (bridges), also acts to pull neighboring doublets closer together. This force resists termination of a sliding episode once initiated, and acts locally to give the population of dynein bridges a type of excitability. In other words, as bridges form, the probability of nearby bridges attaching is increased by a positive feedback exerted through the interdoublet spacing. A conceptual working hypothesis explaining the behavior of cilia and flagella is proposed based on the above concepts. Additionally, the feasibility of this proposed mechanism is demonstrated using a computer simulation. The simulation uses a Monte Carlo‐type algorithm for dynein attachment and adhesive force, together with a geometric evaluation of the t‐force on the key microtubule pairs. This model successfully develops spontaneous oscillations from any starting configuration (including a straight position). It is compatible with the physical dimensions, mechanical properties and bridge forces measured in real cilia and flagella. In operation, it exhibits many of the observed actions of cilia and flagella, most notably wave propagation and the ability to produce both cilia‐like and flagella‐like waveforms. © 1994 Wiley‐Liss, Inc.
Extrusion of rotating microtubules on the dynein‐track from a microtubule‐dynein γ‐complex
Authors: Mimori Y. , Miki‐Noumura T. 
Published: February 2005

AbstractApplying a new in vitro motility assay system for microtubules and 22S dynein, we recently reported on an ATP‐induced extrusion of microtubules from microtubule‐dynein α‐ and β‐complexes [Mimori and Miki‐Noumura, 1994:Cell Motil. Cytoskeleton 27:180–191]. In the present study, we prepared a γ‐complex by copolymerizing porcine brain tubulin and Tetrahymena ciliary 22S dynein, and examined the ATP‐induced microtubule movement from the γ‐complex. The extrusion process appeared quite similar to that of the β‐complex. The sliding velocity was 18.39 ± 2.20 m̈m/sec, which was a value comparable to that of trypsin‐digested flagellar axonemes [Yano and Miki‐Noumura, 1980:J. Cell Sci. 44:169–186]. Higher velocity may be due to a densely arranged dynein‐track with the same polarity, which was detached from the γ‐complex and absorbed in rows on a glass surface of the slide. Sometimes a free‐floating microtubule in the perfusion chamber was observed riding and sliding on the dynein‐track remaining on the slide after extrusion.Unexpectedly, we found that when the front part of the microtubule was fixed to a glass surface, a continuous sliding microtubule at the rear part on the dyneintrack often transformed into a left‐handed helix, and subsequently a twisted helix with several turns. The helix formation may be due to some rigidity in the microtubule and a right‐handed torque component in the sliding force of 22S dynein. The addition of ATP may release some distortion accumulated in the complex structure during copolymerization of tubulin and 22S dynein, inducing reverse rotation of the microtubule. © 1995 Wiley‐Liss, Inc.
Listeria monocytogenes intracellular migration: Inhibition by profilin, vitamin D‐binding protein and DNase I
Authors: Sanger Jean M. , Mittal Balraj , Southwick Frederick S. , Sanger Joseph W. 
Published: February 2005

AbstractInfection of host cells by Listeria monocytogenes results in the recruitment of cytoplasmic actin into a tail‐like appendage that projects from one end of the bacterium. Each filamentous actin tail progressively lengthenes, providing the force which drives the bacterium in a forward direction through the cytoplasm and later results in Listeria cell‐to‐cell spread. Host cell actin monomers are incorporated into the filamentous actin tail at a discrete site, the bacterial‐actin tail interface. We have studied the consequences of microinjecting three different actin monomer‐binding proteins on the actin tail assembly and Listeria intracellular movement. Introduction of high concentrations of profilin (estimated injected intracellular concentration 11–22 m̈M) into infected PtK2 cells causes a marked slowing of actin tail elongation and bacterial migration. Lower intracellular concentrations of two other injected higher affinity monomer‐sequenstering proteins, Vitamin D‐binding protein (DBP; 1–2 m̈M) and DNase I (6–7 m̈M) completely block bacterial‐induced actin assembly and bacterial migration. The onset of inhibition by each protein is gradual (10–20 min) indicating that the mechanisms by which these proteins interfere with Listeria‐induced actin assembly are likely to be complex. To exclude the possibility that Listeria recruits preformed actin filaments to generate the tails and that these monomer‐binding proteins act by depolymerizing such performed actin filaments, living infected cells have been injected with fluorescently labeled phalloidin (3 m̈M). Although the stress fibers are labeled, no fluorescent phalloidin is found in the tails of the moving bacteria. These results demonstrate that Listeria‐induced actin assembly in PtK2 cells is the result of assembly of actin monomers into new filaments and that Listeria's ability to recruit polymerization competent monomeric actin is very sensitive to the introduction of exogenous actin monomer‐binding proteins. © 1995 Wiley‐Liss, Inc.
Cytoskeletal domains in the activated platelet
Authors: Bearer E. L. 
Published: February 2005

AbstractPlatelets circulate in the blood as discoid cells which, when activated, change shape by polymerizing actin into various structures, such as filopodia and stress fibers. In order to understand this process, it is necessary to determine how many other proteins are involved. As a first step in defining the full complement of actin‐binding proteins in platelets, filamentous (F)‐actin affinity chromatography was used. This approach identified >30 different proteins from ADP‐activated human blood platelets which represented 4% of soluble protein. Although a number of these proteins are previously identified platelet actin‐binding proteins, many others appeared to be novel. Fourteen different polyclonal antibodies were raised against these apparently novel proteins and used to sort them into nine categories based on their molecular weights and on their location in the sarcomere of striated muscle, in fibroblasts and in spreading platelets. Ninety‐three percent of these proteins (13 of 14 proteins tested) were found to be associated with actin‐rich structures in vivo.Four distinct actin filament structures were found to form during the initial 15 min of activation on glass: filopodia, lamellipodia, a contractile ring encircling degranulating granules, and thick bundles of filaments resembling stress fibers. Actin‐binding proteins not localized in the discoid cell became highly concentrated in one or another of these actin‐based structures during spreading, such that each structure contains a different complement of proteins. These results present crucial information about the complexity of the platelet cytoskeleton, demonstrating that four different actin‐based structures form during the first 15 min of surface activation, and that there remain many as yet uncharacterized proteins awaiting further investigation that are differentially involved in this process. © 1995 Wiley‐Liss, Inc.
Identification and molecular characterization of a yeast myosin I
Authors: Goodson Holly V. , Spudich James A. 
Published: February 2005

AbstractThe family of myosin motors is comprised of numerous classes distributed among a diverse set of organisms and cell types. We have identified an unconventional myosin gene (MYO3) in the yeast Saccharomyces cerevisiae and show that it is member of a subclass of unconventional myosin proteins originally found only in the amoeboid organisms Dictyostelium and Acanthamoeba. Identification of this protein in these genetically and morphologically divergent organisms suggests that it will be ubiquitous in eukaryotes and that it has a role in the basic functions of the eukaryotic cell. We have constructed a strain of yeast missing 99% of the MYO3 coding sequence. This mutation has no observable phenotypic effect, placing MYO3 into a growing class of yeast genes which are dispensable under laboratory conditions, perhaps due to genetic redundancy. Alignment of MYO3 with other unconventional myosins shows that it shares with a subset of them a previously unrecognized region of homology in the tail; this region falls within a domain identified as important for mediating nonspecific electrostatic interactions with membranes. The existence of this region suggests that it may be involved in mediating specific protein‐protein interactions, possibly helping to localize this myosin to specific membranes or membrane regions. In addition, we show that “classic” myosin I proteins share a region of hyper‐proline‐richness 10 amino acids before the SH3 domain. Proline‐rich regions have recently been implicated as SH3 binding sites, which suggests that this region might be involved with regulating or in other ways interacting with SH3 domains. © 1995 Wiley‐Liss, Inc.
Myosin heavy chain isoform expression in human myometrium: Presence of an embryonic nonmuscle isoform in leiomyomas and in cultured cells
Authors: Cavaillé F. , Fournier T. , Dallot E. , Dhellemes C. , Ferré F. 
Published: February 2005

AbstractWe had previously found no myosin heavy chain (MHC) changes in expression during pregnancy in human myometrium. In the present work, we compared the MHC pattern of expression in normal human myometrium, pregnant and non‐pregnant, to that in benign tumors of the uterine musculature and in cultured myometrial cells. We used a high‐resolution gel electrophoretic system and monoclonal antibodies directed against smooth muscle and nonmuscle MHCs. Smooth muscle MHCs (SM1, 204 kDa, and SM2, 200 kDa, MHCs) and a nonmuscle MHC of 196 kDa (NM MHC) were detected in pregnant and nonpregnant human myometrium. Pregnant myometrium was found to differ from nonpregnant myometrium by its slightly lower content in NM MHC, whereas the ration of SM1/SM2 was equivalent. In leiomyomas and in cultured cells grown from human myometrium explants, SM1, SM2, and NM MHCs were also expressed. In addition, a nonmuscle MHC of 198/200 kDa (SMemb MHC), which was present in a fetal human uterus but not in adult normal tissue, was observed in leiomyomas and in cultured cells. Expression of SM1 and SM2 MHCs was variable in the different leiomyomas studied. In cultured cells, SM1 and SM2 MHC content was low, but it was enhanced by suppression of serum after cell confluency. Present results confirm that pregnancy‐associated smooth muscle cell hypertrophy is not accompanied by major changes in MHCs. In contrast, cell culturing and cell hyperplasia leading to leiomyoma formation induce substantial modifications in MHCs, including the occurrence of a second type of nonmuscle MHC. © 1995 Wiley‐Liss, Inc.
Antibody against ahosphorylated proteins (MPM‐2) recognizes mitotic microtubules in endosperm cells of higher plant haemanthus
Authors: Smirnova E. A. , Cox D. L. , Bajer A. S. 
Published: February 2005

AbstractIn diverse cell types, monoclonal antibody MPM‐2 recognizes a class of phosphorylated proteins related to microtubule organizing centers and abundant during mitosis. We have used this antibody in an attempt to identify the spatial and temporal localization of putative microtubule organizing centers in endosperm cells of the higher plant Haemanthus. Our results show that MPM‐2 recognized epitope is present in interphase cells and enriched in mitotic cells. In interphase the antibody usually stains cytoplasmic granules. During the interphase‐prophase transition immunoreactive material appears in the nucleus, at the nuclear envelope, and in association with microtubules. Concomitantly, we observed an increase of immunoreactivity of the cytoplasm. During mitosis the phosphorproteins recognized by MPM‐2 are detected in the cytoplasm, in association with microtubules of the spindle, the phragmoplast, and in the newly‐formed cell plate. After completion of mitosis, only the cell plate and cytoplasmic granules are MPM‐2 positive. Extraction of the cells with Triton X‐100 prior to fixation removes staining of the cytoplasm by MPM‐2. The detergent resistant immunoreactive material remains associated with surrounding the nucleus microtubules of the prophase spindle, the core of kinetochore fibers, and the phragmoplast. In the phragmoplast, however, segments of microtubules which are distal to the cell plate are depleted of MPM‐2.These data demonstrate that microtubule arrays of endosperm cells are phosphorylated during mitosis. Thus, similar to animal cells, interphase and mitotic microtubules of higher plants have different properties. Additionally, the localization of detergent resistant MPM‐2 antigen points to the difference in microtubule nucleation/organization between higher plant and animal cells.