bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of spindle pushing forces in vivo during anaphase B in the fungus Nectria haematococca
Authors: Aist James R. , Bayles Carol J. 
Published: February 2005

AbstractForces that elongate the spindle during anaphase B of mitosis might be generated in the asteis, in the spindle, or in both. In the fungus Nectria haematococca, it has already been shown that the asters pull on the spindle pole bodies (SPBs) through‐out anaphase B. In this study, we used computerized video motion analysis to characterize brief episodes of spindle bending and straightening to find out if such bending is caused by spindle pushing forces. In three episodes there were two distinct components of spindle bending and straightening: one spanning the entire episode and comprising spindle elongation and another, superimposed on the first, involving a shortening of the distance between the SPBs. In a fourth episode, only spindle elongation was involved. All four spindles elongated rapidly while bending and underwent net growth during the overall bending‐straightening episode at an average rate of 4.2 μm/min. The path of one aster of a fifth mitotic apparatus was blocked by a large, occluding vacuole. This obstacle caused the migration of the mitotic apparatus to stop, resulting in a long (25 sec) episode of spindle curving and bending, usually without any substantial reduction in the distance between the SPBs as well as a marked reduction (from 4.7 to 0.65 μm/min) in the rate of spindle elongation. The results provide evidence that spindle pushing forces are active in vivo during anaphase B in N. haematococca and that they, along with astral pulling forces, help to elongate the spindle at a mostly constant rate. This is the first demonstration of both kinds of spindle elongation forces in the same organism.
A component of the interphase cytoskeleton is cyclically recruited into spindle poles during mitosis
Authors: Leslie Roger J. , Kohler Eric , Wilson Leslie 
Published: February 2005

AbstractDuring the transition from interphase to mitosis, proteins are recruited into forming spindle poles [Leslie, Cell Motil. Cytoskeleton 16:225‐228, 1990]. Antibodies which recognize these recruited components clearly label spindle poles during mitosis but the location and character of such proteins during interphase remain a mystery. Competition assays using an antibody to a recruited spindle pole protein show that in its disperse form the spindle pole protein is a highly insoluble component of the Cytoskeleton which is dispersed to such an extent during interphase that it is difficult to identify by immunolocalization. The function of recruited spindle pole proteins is unknown but the aggregation/dispersion cycle and the antigen are highly conserved, appearing in sea urchin embryos and tissue culture cells.
An automated assay for quantifying the swimming behavior of Paramecium and its use to study cation responses
Authors: Clark Kevin D. , Nelson David L. 
Published: February 2005

AbstractParamecium letraurelia is a ciliated protist that alters its swimming behavior in response to various stimuli. Like the sensory responses of many organisms, these responses in Paramecium show adaptation to continued stimulation. For quantitative studies of the initial response to stimulation. and of the time course of adaptation, we have developed a computerized motion analysis assay that can detect deviations from the normal swimming pattern in a population of cells. The motion of an average of ten cells was quantified during periods ranging from 15 to 60 seconds, with a time resolution of 1/15 seconds. During normal forward swimming, the maximum deviation from a straight‐line path was less than 17°. Path deviations above this threshold value were defined as changes in swimming direction. The percentage of total path time that cells spent deviating from forward swimming was defined as percent directional changes (PDC). This parameter was used to construct dose‐response curves for the behavioral effects of various externally added cations known to induce behavioral changes and also to show the time course of adaptation to a depolarizing K+ stimulus. This assay is a valuable tool for studies of chemoeffectors or mutations that alter the swimming behavior of Paramecium and may also be applicable to other motile organisms.
Neural crest cell galvanotaxis: New data and a novel approach to the analysis of both galvanotaxis and chemotaxis
Authors: Gruler Hans , Nuccitelli Richard 
Published: February 2005

AbstractThe galvanotaxis response of neural crest cells that had migrated out of the neural tube of a 56‐hr‐old quail embryo, onto glass coverslips was observed using time‐lapse video microscopy. These cells exhibit a track velocity of about 7 μm/min and actively translocate toward the negative pole of an imposed DC electric field. This nonrandom migration could be detected for fields as low as 7 mV/mm (0.4 mV/cell lepgth). We find that this directional migration is independent of the speed of migration and have generated a rather simple mathematical equation that fits these data. We find that the number of cells that translocate at a given angle, Φ, with respect to the field is given by the equation N (Φ) = exp(a()0 + a1 cos Φ), where a1 is linearly proportional to the electric field strength for fields less than 390 mV/mm with a constant of proportionality equal to KG, the galvanotaxis constant. We show that KG = (150 mV/mm)−1, and at this field strength the cellular response is approximately half maximal. This approach to cellular translocation data analysis is generalizable to other directed movements such as chemotaxis and allows the direct comparison of different types of directed movements. This analysis requires that the response of every cell, rather than averages of cellular responses, is reported. Once an equation for N(Φ) is derived, several characteristics of the cellular response can be determined. Specifically, we describe (1) the critical field strength (390 mV/mm) below which the cellular response exhibits a simple, linear dependence on field strength (for larger field strengths, an inhibitory constant can be used to fit the data, suggesting that larger field strengths influence a second cellular target that inhibits the first); and (2) the amount of information the cell must obtain in order to generate the observed asymmetry in the translocation distribution (for a field strength of 100 mV/mm, 0.3 bits of information is required).
A quantitative study of the role of F‐actin in producing neutrophil shape
Authors: Watts Raymond G. , Crispens Marta A. , Howard Thomas H. 
Published: February 2005

AbstractNeutrophils change shape from round to polar and sequentially polymerize/depolymerize actin following chemotactic peptide activation in suspension. To study the relationship between changes in F‐actin content and shape we altered the kinetics/extent of actin polymerization and depolymerization with tBOC peptide, cytochalasin D (CD), and low‐dose FMLP, and determined the effect of these alterations on the temporal sequence of changes in neutrophil shape. F‐actin was measured by FACS analysis of NBDphallacidin‐stained cells and expressed as relative fluorescent intensity (RFI) compared to control (RFI = 1.00). Shape was determined by scanning electron microscopy. FMLP causes serial polymerization/depolymerization of actin (RFI = 1.00 ± 0.04, 1.60 ± 0.21, 1.10 ± 0.18, and 1.05 ± 0.14) associated with four distinct shapes (round‐smooth, round‐ruffled, blebbed, and polar) noted at 0, 30, 90, 300 sec respectively. Since blebbed and polar shapes appear concurrent with depolymerization and following polymerization, we determined whether depolymerization is required for polarization of cells. The kinetics of depolymerization were: (1) accelerated by tBOC addition at 45 sec, and (2) slowed by high concentrations of FMLP (>10‐7 M) (300 sec RFI = 1.46). Neither change altered the time course of shape change. To determine whether duration of actin polymerization defines shape, polymerization was halted by addition of tBOC at 5, 10, 20, 30 sec after FMLP to block actin polymerization and shape was monitored at 300 sec. TBOC added 5–20 sec after FMLP limited neutrophil shape change to the blebbed form, while tBOC addition 30 sec following FMLP resulted in a polar shape at 300 sec. To determine whether the extent of actin polymerization affects the shape change sequence, polymerization was limited by (1) inhibition of polymerization with CD, (2) exposure of cells to low concentrations of FMLP ( < 10‐9 M), and (3) interruption of polymerization with tBOC. Actin polymerization to RFI < 1.35‐fold basal results in blebbed shape; polymerization > 1.35‐fold basal yields polar shape. The data show: (1) the human neutrophil demonstrates intermediate shapes when activated by chemotactic peptide, (2) depolymerization of F‐actin does not determine shape, and (3) blebbed shape appears when actin polymerizes for >5 sec; polar shape with polymerization ≥30 sec to RFI > 1.35‐fold basal. The data suggest actin polymerization is required for, and extent of polymerization determines, the shape of human neutrophils.
Metabolic regulation in mammalian sperm: Mitochondrial volume determines sperm length and flagellar beat frequency
Authors: Cardullo Richard A. , Baltz Jay M. 
Published: February 2005

AbstractIn the absence of glycolytic support, mammalian sperm derive their energy for motility from a densely packed array of mitochondria at the base of the flagellum known as the midpiece. Using data on the morphometric dimensions of over 200 mammalian species, we found that an allometric relationship exists between midpiece length (Lm) and flagellum length (Lf). Specifically, the length of the midpiece varies approximately as the 3/2 power of the flagellar length although the proportionality constant is different for eutherian and marsupial sperm. In contrast, when we corrected for the fraction of the midpiece that was taken up by mitochondria, a single linear correlation between mitochondrial volume and flagellar length for all mammals was found. These allometric relationships were used along with basic flagellar hydrodynamic theories to establish a unifying equation that predicted flagellar frequencies for any mammalian sperm between 40 μm and 200 μm in length. These findings imply that, at least in mammals, the mechanisms for energy production and dissipation in sperm flagella are highly conserved.
Ultrastructure of the human erythrocyte cytoskeleton and its attachment to the membrane
Authors: Ursitti Jeanine A. , Pumplin David W. , Wade James B. , Bloch Robert J. 
Published: February 2005

AbstractWe attached paraformaldehyde‐fixed human erythrocyte ghosts to coated coverslips and sheared them to expose the cytoskeleton. Quick‐freeze, deep‐etch, rotary‐replication, or tannic acid/osmium fixation and plastic embedding revealed the cytoskeleton as a dense network of intersecting straight filaments. Previous negative stain studies on spread skeletons found 5–6 spectrin tetramers intersecting at each actin oligomer, with an estimated 250 such intersections/μm2 of membrane. In contrast, we found 3–4 filaments at each intersection and ∼400 intersections/μm2 of membrane. Immunogold labeling verified that the filaments were spectrin, but their lengths (29–37 nm) were approximately one‐third that of extended spectrin dimers. The length and diameter of the filaments were sufficient to accommodate spectrin dimers, but not spectrin tetraments. Our results suggest that, in situ, spectrin dimers may associations as hexamers and octamers, rather than tetramers. We present several explanations that can reconcile our observations on intact cytoskeletons with previous reports on spread material.Extracting sheared ghosts with solutions of low ionic strength removed the cytoskeleton to reveal projections from the cytoplasmic surface of the membrane. These projections contained band 3, as shown by immunogold labeling, and they aggregated to a similar extent as intramembrane particles (IMP) when the cytoskeleton was removed, suggesting a direct relationship between these structures. Quantification indicated a stoichiometry of 2 IMP for each cytoplasmic projection. Cytoplasmic projections presumably contain other proteins besides band 3 since further treatment with high ionic strength solutions extracts peripheral proteins and reduces the diameter of projections by ∼3 nm.
F‐actin bundling protein from Physarum polycephalum: Purification and its capacity for co‐bundling of actin filaments and microtubules
Authors: Itano Naoki , Hatano Sadashi 
Published: February 2005

AbstractAn F‐actin bundling protein was isolated and purified from plasmodium of Physarum polycephalum. The F‐actin bundling protein in Physarum extract was passed through a DEAE‐cellulose column. After the protein in the fraction was treated with 6 M urea, it was purified by gel filtration on Sephacryl S‐300 HR followed by chromatography on CM‐Toyopearl (cation exchange) in the presence of 6 M urea. The purified protein gave a single band on SDS‐PAGE, and the molecular weight was estimated to be 52,000. This F‐actin bundling protein is referred to as the 52 kDa protein.Interestingly, the 52 kDa protein also induced bundling of microtubules. The formation of F‐actin and microtubule bundles was Ca2+‐insensitive, but depended on the salt concentration. Each bundle formed at NaCl concentrations less than 0.1 M. The 52 kDa protein cross‐reacted with monoclonal antibody raised against a HeLa 55 kDa protein (an F‐actin bundling protein from HeLa cells) (Yamashiro‐Matsumura and Matsumura: J. Biol. Chem. 260:5087‐5097, 1985).When the 52 kDa protein was added to a mixture of actin filaments and microtubules, co‐bundles composed of both filaments formed. This is the first reported example in which an F‐actin bundling protein induced co‐bundling of actin filaments and microtubules.
Substrate‐bound factors stimulate engorgement of growth cone lamellipodia during neurite elongation
Authors: Burmeister Donald W. , Rivas Rodolfo J. , Goldberg Daniel J. 
Published: February 2005

AbstractThe surfaces on which neurons grow greatly affect neurite elongation, but it is unclear how substrates influence the events within the growth cone that bring about elongation. Neurite elongation by Aplysia californica neurons in culture occurs through a series of transformations of the structures of the growth cone (Goldberg and Burmeister, J. Cell Biol., 103:1921‐1931, 1986). The growth cone produces actin‐rich protrusions, veils, and lamellipodia, which can then mature into the central body of the growth cone through the net advance of microtubules and membranous organelles from contiguous central regions, a process called “engorgement.” Aplysia neurons form growth cones on poly‐l‐lysinetreated substrates, but their rate of neurite elongation is greatly enhanced on substrates additionally exposed to Aplysia hemolymph. The acute application of hemolymph to slowly growing neurites brings about a rapid acceleration of neurite elongation and engorgement. The enhancement of engorgement was effected with material eluted from hemolymph‐treated substrates and was not seen when hemolymph was added to neurons cultured on hemolymph‐treated substrates inactivated by exposure to UV radiation. Thus, we conclude that the rapid acceleration of engorgement caused by hemolmph is, in large part, a substrate‐mediated effect. We propose that extracellular substrate molecules can modulate the rate of neurite growth through the regulation of the engorgement of lamellipodia.The microtubule disrupters colcemid and nocadazole inhibit the advance of vesicular elements into the lamellipodia following hemolymph treatment, but taxol, which promotes the polymerization and stabilization of microtubules, does not itself enhance engorgement. The microfilament disrupter cytochalasin B, however, stimulates engorgement. Our results suggest that regulating the resistance of the peripheral actin meshwork to penetration by microtubules and vesicles may be a mechanism by which substrate‐attached molecules regulate neurite advance.
Microtubule sliding in flagellar axonemes of Chlamydomonas mutants missing inner‐ or outer‐arm dynein: Velocity measurements on new types of mutants by an improved method
Authors: Kurimoto Eiji , Kamiya Ritsu 
Published: February 2005

AbstractTo help understand the functional properties of inner and outer dynein arms in axonemal motility, sliding velocities of outer doublets were measured in disintegrating axonemes of Chlamydomonas mutants lacking either of the arms. Measurements under improved solution conditions yielded significantly higher sliding velocities than those observed in a previous study [Okagaki and Kamiya, 1986, J. Cell Biol. 103:1895‐1902]. As in the previous study, it was found that the velocities in axonemes of wild type (wt) and a mutant (oda1) missing the outer arm differ greatly: 18.5 ± 4.1 μm/sec for wt and 4.4 ± 2.3 μm/sec for oda1 at 0.5 mM Mg‐ATP. In contrast, axonemes of two types of mutants (ida2 and ida4) that lacked different sets of two inner‐arm heavy chains displayed velocities almost identical with the wild‐type velocity. Moreover, axonemes of a non‐motile double mutant ida2 × ida4 underwent sliding disintegration at a similar high velocity, although less frequently than in axonemes of single mutants. These observations support the hypothesis that the inner and outer dynein arms in disintegrating axonemes drive microtubules at different speeds and it is the faster outer arm that determines the overall speed when both arms are present. The inner arm may be important for the initiation of sliding. The axoneme thus appears to be equipped with two (or more) types of motors with different intrinsic speeds.
The dynamic instability of microtubules is not modulated by α‐tubulin tyrosinylation
Authors: Idriss Haitham , Stammers David K. , Ross Carl K. , Burns Roy G. 
Published: February 2005

AbstractThe tyrosinylation of chick brain α‐tubulin and the effects of the tyrosinylation status on the assembly and dynamic instability of chick brain MAP2:tubulin microtubule protein have been examined. Each of the eight major α‐isotypes can be tyrosinylated in vitro, irrespective of whether a C‐terminal tyrosine is genetically encoded. The extent of tyrosinylation is however limited to ≏ 0.3 mol.mol−1. The tyrosinylation status (0 vs. 0.3 mol.mol−1) has no effect on either the assembly kinetics of chick brain microtubule protein or on the rate of length redistribution following assembly and shearing. It is therefore unlikely that the tyrosinylation status directly affects the intrinsic stability of assembled microtu‐bules since the rate of length redistribution is both a sensitive assay and a function of the kinetic parameters governing dynamic instability.
Epitope mapping of monoclonal antibodies against caldesmon and their effects on the binding of caldesmon to Ca++/calmodulin and to actin or actin‐tropomyosin filaments
Authors: Lin Jim Jung‐Ching , Davis‐Nanthakumar Elizabeth J. , Jin Jian‐Ping , Lourim David , Novy Robert E. , Lin Jenny Li‐Chun 
Published: February 2005

AbstractThe effects of monoclonal anti‐caldesmon antibodies, C2, C9, C18, C21, and C23, on the binding of caldesmon to F‐actin/F‐actin‐tropomyosin filaments and to Ca++/calmodulin were examined in an in vitro reconstitution system. In addition, the antibody epitopes were mapped by Western blot analysis of NTCB (2‐nitro‐5‐thiocyanobenzoic acid) and CNBr (cyanogen bromide) fragments of caldesmon. Both C9 and C18 recognize an amino terminal fragment composed of amino acid residues 19 to 153. The C23 epitope lies within a fragment ranging from residues 230 to 386. Included in this region is a 13‐residue repeat sequence. Interestingly this repetitive sequence shares sequence similarity with a sequence found in nuclear lamin A, a protein which is also recognized by C23 antibody. Therefore, it is likely that the C23 epitope corresponds to this 13‐residue repeat sequence. A carboxyl‐terminal 10K fragment contains the epitopes for antibodies C2 and C21. Among these antibodies, only C21 drastically inhibits the binding of caldesmon to F‐actin/F‐actin‐tropomyosin filaments and tc Ca++/calmodulin. When the molar ratio of monoclonal antibody C21 to caldesmon reached 1.0, a maximal inhibition (90%) on the binding of caldesmon to F‐actin filaments was observed. However, it required double amounts of C21 antibody to exhibit a maximal inhibition of 70% on the binding of caldesmon to F‐actin‐tropomyosin filaments. These results suggest that the presence of tropomyosin in F‐actin enhances caldesmon's binding. Furthermore, C21 antibody also effectively inhibits the caldesmon binding to Ca++/calmodolin. The kinetics of C21 inhibition on caldesmon's binding to Ca++/calmodulin is very similar to the inhibition obtained by preincubation of caldesmon with free Ca++/calmodulin. This result suggests that there is only one Ca++/calmodulin binding domain on caldesmon and this domain appears to be very close to the C21 epitope. Apparently, the Ca++/calmodulin‐binding domain and the actin‐binding domain are very close to each other and may interfere with each other. In an accompanying paper, we have further demonstrated that microinjection of C21 antibody into living chicken embryo fibroblasts inhibit intracellular granule movement, suggesting an in vivo interference with the functional domains [Hegmann et al., 1991: Cell Motil. Cytoskeleton 20:109–120].
Post‐translational incorporation of actin into myofibrils in vitro: Evidence for isoform specificity
Authors: Peng Isaac , Fischman Donald A. 
Published: February 2005

AbstractThe incorporation of actin into myofibrils has been examined in a cell‐free system [Bouché et also Journal of Cell Biology 107:587–596, 1988; Goldfine et all Cellular and Molecular Biology of Muscle Development, 1989]. Actin was translated in a reticulocyte lysate in the presence of 35S‐methionine (35S‐actin) or purified from muscle and labeled with fluorescein‐5‐isothiocyanate (FITC‐actin). Myofibrils were incubated with either 35S‐actin or FITC‐actin and then analyzed by gel electrophoresis or fluorescence microscopy. When myofibrils were incubated with FITC‐actin monomer in the reticulocyte lysate buffer, strong fluorescent labeling was observed in Z‐band regions and less so in I‐bands. No fluorescence was detected in non‐overlap regions of A‐bands. Confocal microscopic analysis of these myofibrils indicated that FITC‐actin was distributed evenly across the diameter of the myofibrils. These observations suggest that actin incorporation in the reticulocyte lysate buffer occurred at sites in the sarcomere which contain actin. In contrast, FITC‐actin showed a variety of non‐physiological incorporation patterns when incubated with myofibrils in the presence of an isotonic buffer (I‐buffer). However, when ATP was added to I‐buffer, FITC‐actin showed a pattern of incorporation into myofibrils similar to that seen in the reticulocyte lysate buffer. Immunoblots indicated that actin of native size was released from myofibrils during incubation in the reticulocyte lysate buffer. No actin release was detected when the myofibrils were incubated in I‐buffer lacking ATP. We used this system to compare the incorporation of actin isoforms into myofibrils. Both α‐ and β‐actins exhibited incorporation into the myofibrils but there was a three‐fold greater incorporation of the α isoform. We propose that the differential affinities of actin isoforms for myofibrils and other cytoskeletal structures could provide a mechanism for actin isoform targeting within the cytoplasm.