bluuebunny/crossref_metadata_2025_split huggingface dataset:
The centrosomal cycle: Visualization in PtK cells by a monoclonal antibody to a centrosomal 32 kd protein
Authors: Joswig Gaby , Petzelt Christian 
Published: February 2005

AbstractA monoclonal antibody prepared against a crude centrosome fraction from PtK cells reacts exclusively with centrosomes. Using Western blotting techniques, the antigen was identified as a protein with a molecular weight of 32 kd. Using this probe it is possible to follow pronounced shape changes of the centrosome through the cell cycle and to study its replication. When the microtubular cytoskeleton is removed by nocodazole, neither the shape nor the three‐dimensional organization of the centrosome inside the cell are altered. Moreover, in spite of the cell cycle arrest caused by nocodazole, the centrosomal cycle proceeds, thus indicating its independence from the intact cytoskeleton and supporting its role as a semiautonomous organelle. On the basis of these results we hypothesize that the centrosome has two distinct functions: in the non‐growing compact state during mitosis the centrosome serves as the pole organizer and during interphase it functions as the “cytocenter”.
Nucleotide specificity for the bidirectional transport of membrane‐bounded organelles in isolated axoplasm
Authors: Leopold Philip L. , Snyder Robert , Bloom George S. , Brady Scott T. 
Published: February 2005

AbstractVideo microscopy of isolated axoplasm from the squid giant axon permits correlated quantitative analyses of membrane‐bounded organelle transport both in the intact axoplasm and along individual microtubules. As a result, the effects of experimental manipulations on both anterograde and retrograde movements of membrane‐bounded organelles can be evaluated under nearly physiological conditions. Since anterograde and retrograde fast axonal transport are similar but distinct cellular processes, a systematic biochemical analysis is important for a further understanding of the molecular mechanisms for each. In this series of experiments, we employed isolated axoplasm of the squid to define the nucleoside triphosphate specificity for bidirectional organelle motility in the axon. Perfusion of axoplasm with 2–20 mM ATP preserved optimal vesicle velocities in both the anterograde and retrograde directions. Organelle velocities decreased to <50% of optimal values when the axoplasm was perfused with 10–20 mM UTP, GTP, ITP, or CTP with simultaneous depletion of endogenous ATP with hexokinase. Under the same conditions, TTP and ATP‐γ‐S were unable to support significant levels of transport. None of the NTPs tested had a differential effect on anterograde vs. retrograde movement of vesicles. Surprisingly, several inconsistencies were revealed when a comparison was made between these results and nucleoside triphosphate specificities that have been reported for putative organelle motors by using in vitro assays. These data may be used in conjunction with data from well‐defined in vitro assays to develop models for the molecular mechanisms of axonal transport.
Ultrastructural cytoskeleton alterations and modification of actin expression in the NIH/3T3 cell line after transformation with Ha‐ras‐Activated oncogene
Authors: Lombardi Luciano , Ballinari Dario , Bongarzone Italia , Migliari Marco , Mondellini Piera , Traversai Catia , Modina Silvia 
Published: February 2005

AbstractCytoskeleton alterations of NIH/3T3 fibroblast monolayers transfected with Haras‐activated oncogene were studied by immunofluorescence, immunoelectron microscopy, and immunoelectrophoretic analysis of actin isoforms. Transformation foci were found to consist of cells with a round shape and rare stress fibers that spread sparsely, forming rare focal contacts and fibronexuses. The loss of stress fibers in transformed cells was confirmed by staining with rhodamine‐phalloidin and with a fluorescinated anti‐non‐muscle cell actin antibody. The transformed cells were anchored to the substrate prominently by filaments that contained fibronectin, as showed by immunoelectron microscopy. A down‐regulation of α‐actin isoform was observed by immunofluorescence and immunoblotting analysis using a specific monoclonal antibody. The diffuse distribution of α‐actin, lacking a specific association with stress fibers, challenges the hypothesis of a connection between α‐actin down‐regulation and stress fiber loss.
Formation, transport, contraction, and disassembly of stress fibers in fibroblasts
Authors: Giuliano Kenneth A. , Lansing Taylor D. 
Published: February 2005

AbstractSwiss mouse 3T3 fibroblasts grown on a solid substrate in the presence of 10% serum exhibit cell movement, organelle transport, and cytokinesis. When the serum concentration in the culture medium is decreased to 0.2% for 48 h the serum‐deprived cells virtually stop locomoting, spread, decreased organelle transport, and exhibit extensive arrays of stress fibers that are visible with video‐enhanced differential interference contrast microscopy and that also incorporate fluorescent analogs of actin and conventional myosin (myosin II). The stress fibers form in a constitutive mannet at the cytoplasm‐membrane interface, transport toward the nucleus, and then disappear. The rate of transport of these fibers is quite heterogeneous with average rates in the range of 10–20 μm/h. When serum‐deprived cells are stimulated with mitogens such as 10% serum or 10 nM thrombin, many of the stress fibers immediately begin to shorten, suggesting a contraction. The rate of shortening is approximately two orders of magnitude slower than that of unloaded smooth muscle cells. The fiber shortening is often accompanied by retraction of the edges of the cell and continues for at least the 1st hour post‐stimulation.
Molecular cloning and expression of sea urchin embryonic ciliary dynein β heavy chain
Authors: Foltz Kathleen R. , Asai David J. 
Published: February 2005

AbstractThe determination of the structure and the expression of dynein during embryonic development are central to the understanding of dynein function. As an important first step toward these objectives, cDNAs encoding portions of sea urchin ciliary dynein were identified by antibody screening of a sea urchin cDNA expression library. Bacause of the complete lack of protein sequence data, it was first necessary to prove the identity of the dynein cDNAs. Of the five cDNA inserts initially cloned, one, designated P72A1, was characterized extensively. Four independent criteria demonstrated that P72A1 encoded a portion of a dynein heavy chain. (1) The β‐galactosidase‐P72A1 fusion protein affinity‐purified dynein‐specific antibodies from crude antiserum. (2) Two other antisera to dynein, raised independently of the antiserum used to screen the cDNA library, reacted with the fusion protein. (3) A new antiserum raised against the fusion protein reacted with authentic dynein heavy chain on Western blots and stained embryonic cilia by indirect immunofluorescence microscopy. (4) Two new antisera, elicited against opposite ends of the P72A1 open reading frame, each reacted with authentic dynein heavy chain protein. Western blot analyses of dissociated dynein heavy chains revealed that P72A1 encoded a portion of the β heavy chain. Epitope mapping experiments confirmed the identity of P72A1 as part of the βheavy chain and also demonstrated that P72A1 encoded epitopes of the carboxyl‐terminal fragment B domain of the dynein β heavy chain. Northern blot analyses of poly(A)+ RNA revealed that P72A1 hybridized with a large RNA species ca. 12.5 kb in length. The dynein mRNA concentration increased during embryonic development. Dot blot analyses of RNA isolated at various times after embryo deciliation demonstrated that the dynein β heavy chain mRNA accumulated rapidly in response to deciliation. The accumulation was similar to but not identical with the induction of tubulin mRNA in response to the same stimulus.
Role of the cytoskeleton during injury‐induced cell migration in corneal endothelium
Authors: Gordon Sheldon R. , Staley Carol A. 
Published: February 2005

AbstractThe role of microfilaments and microtubules during injury‐induced cell migration of corneal endothelial cells in situ along their natural basement membrane has been investigated using organ culture. In the noninjured tissue, actin is localized at or near the plasma membrane, whereas tubulin is observed as a delicate lattice pattern throughout the cytoplasm. Twenty‐four hours after a circular freeze injury, cells surrounding the wound area extend processes into this region. Fluorescent microscopy using phallotoxins and anti‐tubulin antibodies demonstrated the presence of stress fibers and microtubule reorganization within these cells. Between 24 and 48 h post‐injury endothelial cells move into the wound region, and by 48 h, the injury zone is repopulated and the monolayer is becoming resstablished. When injured corneas are placed in media containing 5 × 10−7 M cytochalasin B, endothelial cell migration occurs: but it is slow, and wound closure is not complete even by 72 h. In contrast, when tissues are cultured in the presence of 10−8 M colchicine, cell movement is greatly reduced, complete wound closure does not occur, and endothelial cells at the wound edge fail to display extensions typical of migrating cells. Furthermore, when injured endothelia are exposed to 0.05 μg/ml of actinomycin D for 15 min within the first hour after injury and transferred back into culture media lacking the drug for the duration of the experiment, migration does not occur and the wound persists. These actinomycin D treated cells remain viable as shown by their ability to incorporate 3H‐uridine and 3H‐thymidine. Fluorescence microscopy of actinomycin D treated tissues revealed the presence of stress filaments but disorganized microtubule patterns. Interestingly, 24 h after injury, if the tissue is exposed to actinomycin D, even for periods of up to 1 h, migration is not inhibited. Our results indicate that injury‐induced endothelial cell movement appears to be more dependent on microtubule than microfilament reorganization and may require a critical timing of macromolecular synthesis.
Cytoskeletal dynamics in rabbit synovial fibroblasts: I. Effects of acrylamide on intermediate filaments and microfilaments
Authors: Aggeler Judith , Seely Keith 
Published: February 2005

AbstractRabbit synovial fibroblasts respond to changes in cell shape and cytoskeletal architecture by altering specific gene expression. We have tested the ability of acrylamide, a neurotoxin that alters the distribution of intermediate filaments in cultured PtKl cells, to induce metalloprotease expression in synovial fibroblasts. Cells treated with 2–20 mM acrylamide for 5 to 24 h underwent shape changes similar to cells treated with the tumor promoter phorbol myristate acetate. Intermediate filaments visualized with anti‐vimentin antibodies did not collapse into a perinuclear cap in these rounded cells, but were still present in the extended cell processes. Unexpectedly, when actin was visualized in acrylamide‐treated cells, extensive dissociation and clumping of microfilaments was observed. Concentrations of acrylamide > 10 mM were cytotoxic, but cells recovered completely after 24 h incubation with 5 mM acrylamide. Like other agents that alter cell shape and actin distribution in synovial fibroblasts, acrylamide also induced expression of the secreted metalloprotease collagenase. Although some recent evidence suggests that acrylamide may be able to exert its collagenase‐inducing effects extra‐cellularly, perhaps through transmembrane matrix receptors, our observation that this neurotoxin dramatically alters protein synthesis in synovial fibroblasts suggests that direct effects on cell metabolism may also play a role in acute acryl‐amide intoxication.
The cytoskeletal system of mammalian primitive erythrocytes: Studies in developing marsupials
Authors: Cohen William D. , Cohen Marion F. , Hugh Tyndale‐Biscoe C. , Vandeberg John L. , Ralston Gregory B. 
Published: February 2005

AbstractSeeking to resolve conflicting literature on cytoskeletal structure in mammalian “primitive” generation erythrocytes, we have utilized the circulating blood of developing marsupials. In young of the Tammar Wallaby (Macropus eugenii) and the Gray Short‐tailed Opossum (Monodelphis domestica), relatively large, nucleated primitive erythrocytes constituted nearly 100% of the circulating population of birth (= day 0) and in fetuses (Tammar) several days before birth. These cells were discoidal or elliptical, and flattened except for a nuclear bulge. Their cytoskeletal system, consisting of a marginal band of microtubules enclosed within a cell surface‐associated network (membrane skeleton), closely resembled that of non‐mammalian vertebrate erythocytes. By day 2 or 3, much smaller anucleate erythrocytes of “definitive” morphology, lacking marginal bands, appeared in abundance. These accounted for >90% of the circulating population of both species by day 6–8. Non‐nucleated erythrocytes of a different type, constituting 1–6% of the cells in most blood samples up to day 7, were identified as anucleate primitives on the basis of size, shape, and presence of a marginal band. Thus, loss of erythrocyte nuclei in mammals appears to begin earlier than generally recognized, i.e., in the primitive generation. Counts of these anucleate primitives in young of various ages implicated nucleated primitives as their probable source. Pointed erythrocytes, occasionally found in younger neonates of both species, occurred in greatest number in fetuses (Tammar) prior to birth. This is in accord with previous work on non‐mammalian vertebrates suggesting that such cells are morphogenetic intermediates. The results confirm the long‐suspected similarity between mammalian primitive erythrocytes and the nucleated erythrocytes of all non‐mammalian vertebrates.
On metachronism in ciliary systems: A model describing the dependence of the metachronal wave properties on the intrinsic ciliary parameters
Authors: Gheber Larisa , Priel Zvi 
Published: February 2005

AbstractA mathematical model is proposed to explain the dependence of the direction and the length of the metachronal wave on parameters that characterize the ciliary beat, the dimensions of the cilia, and the geometry of their arrangement on the ciliated surface. The metach/onal wave is decomposed into two mutually perpendicular components, which are chosen in such a way that the direction of one of them is in the direction of the effective stroke. The magnitudes of the two components are determined by using the concept of the time of delay between adjacent cilia. The properties of the metachronal wave are then calculated as a function of the ciliary parameters.The results obtained with the present model predict that the direction of the wave propagation is strongly dependent on the type of metachronism in the direction of the effective stroke and the polarization in time and in space of the ciliary beat. The metachronal wavelength is found to depend on four parameters: the ciliary length, the angle of the arc projected on the cell surface by the ciliary tip during the recovery stroke, the degree of asymmetry of ciliary beat, and the portion of the cycle occupied by the pause. The metachronal wavelength is also found to be only weakly dependent on the ciliary frequency.At this stage there exists relatively little experimental information with which t o characterize fully the metachronal properties of ciliary systems. Even when only partial information exists, the model allows prediction, to within a certain range, of the direction of the wave propagation. It also suggests a possible mechanism for the influence of changes in environmental conditions on wave direction and wavelength. In severalcases in which full information does exist, good agreement between the experimental findings and the predictions of the model is found. According to this model it will be worthwhile to invest more effort in measuring the time and space polarization of ciliary beating and the times of delay between cilia.
Quantitative relationships between single‐cell and cell‐population model parameters for chemosensory migration responses of alveolar macrophages to C5a
Authors: Farrell Brian E. , Daniele Ronald P. , Lauffenburger Douglas A. 
Published: February 2005

AbstractPhenomenological parameters from a mathematical model of cell motility [1] are used to quantitatively characterize chemosensory migration responses of rat alveolar macrophages migrating to C5a in the linear under‐agarose assay, simultaneously at the levels of both single cells and cell populations. This model provides theoretical relationships between single‐cell and cell‐population motility parameters. Our experiments offer a critical test of these theoretical linking relationships, by comparison of results obtained at the cell population level to results obtained at the single‐cell level.Random motility of a cell population is characterized by the random motility coefficient, μ (analogous to a particle diffusion coefficient), whereas single‐cell random motility is described by cell speed, s, and persistence time, P (related to the period of time that a cell moves in one direction before changing direction). Population chemotaxis is quantified by the chemotactic sensitivity, χo, which provides a measure of the minimum attractant gradient necessary to elicit a specified chemotactic response. Single‐cell chemotaxis is characterized by the chemotactic index, CI, which ranges from 0 for purely random motility to 1 for perfectly directed motility. Measurements of cell number versus migration distance were analyzed in conjunction with the phenomenological model to determine the population parameters while paths of individual cells in the same experiment were analyzed in order to determine the single‐cell parameters.The parameter μ shows a biphasic dependence on C5a concentration with a maximum of 1.9 × 10−8 cm2/sec at 10−11 M C5a and relative minima of 0.86 × 10−8 cm2/sec at 10−7 M C5a and 1.1 × 10−8 cm2/sec in the absence of C5a; s and P remain fairly constant with C5a concentration, with s ranging from 2.1 to 2.5 μm/min and P varying from 22 to 32 min. χo is equal to 1.0 × 10−6 cm/receptor for all C5a concentrations tested, corresponding to 60% correct orientation for a difference of 500 bound C5a receptors across a 20 μm cell length. The maximum CI measured was 0.2.Values for the population parameters μ and χo were calculated from single‐cell parameter values using the aforementioned theoretical linking relationships. The values of μ and χo calculated from single‐cell parameters agreed with values of μ and χo determined independently from population migrations, over the full range of C5a concentrations, confirming the validity of the linking equations. Experimental confirmation of such relationships between single‐cell and cell‐population parameters has not previously been reported.
Immunocytochemical identification of cytoskeletal linkages to smooth muscle cell nuclei and mitochondria
Authors: Stromer Marvin H. , Bendayan Moise 
Published: February 2005

AbstractIn avian smooth muscle cells, desmin‐containing intermediate filaments (IFs) are a prominent component of the cytoskeleton and are readily seen in several domains, inclu‐ding the axial intermediate filament bundle (IFB). Both the nucleus and some of the mitochondria are partly surrounded by elements of the IFB. By using anti‐desmin and protein‐A‐colloidal gold labeling, we have identified intermediate filaments that form linkages with the nuclear envelope and with mitochondria. These linkage regions seem to occupy a proportionately greater part of the mitochondrial surface than of the nuclear envelope. The existence of these linkages in smooth muscle cells is consistent with results that support similar linkages to mitochondria and other cellular structures in various cells that contain either vimentin or keratin IFs. These linkages could functionally restrain or assist in homeostatically restoring organelles to their normal position after the rearrangement that accompanies the substantial shortening of smooth muscle cells.
Distribution of microtubules containing post‐translationally modified α‐tubulin during drosophila embryogenesis
Authors: Warn R. M. , Harrison A. , Planques V. , Robert‐Nicoud N. , Wehland J. 
Published: February 2005

AbstractThe distribution of microtubules (MTs) enriched in detyrosinated α‐tubulin (Glu‐tubulin) was studied in Drosophila embryos by immunofluorescence micro‐scopy by using a monoclonal antibody (ID5) which was raised against a 14‐residue synthetic peptide spanning the carboxyterminal sequence of Glu‐tubulin (Wehland and Weber: J. Cell Sci. 88:185‐203, 1987). While all MT arrays contained tyrosinated α‐tubulin (Tyr‐tubulin), MTs rich in Glu‐tubulin were not found during early stages of development even by using an image intensification camera. Elevated levels of microtubular Glu‐tubulin were first detected after CNS condensation in neurone processes. In addition, sperm tails, which remained remarkably stable inside the embryo until late stages of development, were decorated by ID5. This was in marked contrast to the distribution of microtubule arrays containing acetylated α‐tubulin, which could already be detected during the cellular blastoderm stage. Additional experiments with taxol suggested that the absence of MTs rich in Glu‐tubulin during early stages of development was not due to the rapid turnover rate of MTs, which would be too fast for α‐tubulin to be detyrosinated. The possible significance of the differential detyrosination and acetylation of microtubules during development is discussed.
Identification and localization of proteins immunologically related to intermediate filament proteins in sea urchin eggs and embryos
Authors: St‐Pierre Johanne , Dufresne Louise 
Published: February 2005

AbstractSea urchin spermatozoa, eggs, and embryos were labeled with the universal antibody against the intermediate filament proteins (anti‐IFA) described by Pruss et al. [Cell 27:419–428, 1981] and with anti‐beta‐tubulin. Localization of these antibodies was by indirect immunofluorescence microscopy. Cytoskeleton of unfertilized eggs, prepared according to a procedure adapted from Kane [Exp. Cell Res. 162:495–506, 1986] or as described by Dufresne et al. [Biochem. Cell Biol. 66:780–791, 1988], and reacted with the anti‐IFA demonstrate a uniformly stained background except for the nuclear areas, which appear as dark rings. During the first cell cycle, the anti‐IFA staining pattern coincides with that of spindle‐associated tubulin but not with the cortical pattern of microtubules. Swimming embryos reacted with the anti‐IFA show a labeling located on the cilia and within the cytoplasm of each individual cell of the larva. In spermatozoa, the labeling occurs all along the flagellae. Immunoblots of proteins from eggs and embryos reveal one major protein of 117 kDa and sometimes a component of 66 kDa, both of which cosediment with tubulin during the isolation procedure of microtubules described by Vallee and Bloom [Proc. Natl. Acad. Sci. USA 80:6259–6263, 1983]. These data show that proteins homologous to the intermediate filament proteins reported in vertebrate cells are present in both gametes of sea urchins. The specific localization ofthese proteins in the spindle, the flagella, and the cilia suggest that they may play a significant role in the organization and function of the microtubular lattice of the spermatozoa and of the embryo.