bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of fluorescently derivatized bovine tau protein and its localization and functions in cultured Chinese hamster ovary cells
Authors: Lu Qun , Wood John G. 
Published: February 2005

AbstractBovine brain tau protein was tagged with the fluorescent dye 5 (and 6)‐carboxy‐x‐rhodamine‐succinimidyl ester and the functional properties of the fluorescent analog were tested in vitro by kinetic measurement and SDS gel electrophoresis. X‐rhodamine tau was competent to bind to microtubules and promote microtubule assembly in vitro. Labeled tau was further characterized by microinjection of cultured Chinese hamster ovary (CHO) cells to study its intracellular distribution and potential new functions. X‐rhodamine tau incorporated rapidly into centrosomes within seconds after microinjection. It distinctly labeled the microtubule network as early as 5 to 10 minutes following microinjection. In addition, X‐rhodamine tau was transported into the nucleus and labeled the nucleolus specifically. Double labeling of the injected cells with DiC6(3) indicated that in some cases, fluorescent tau may associate with the endoplasmic reticulum. The concentrations of injected X‐rhodamine tau ranged from 1.7 to 5.0 mg/ml, yet distinct bundling of microtubules was not observed. Studies of nocodazole effects on the microtubules established that X‐rhodamine tau stabilized microtubules against depolymerization conditions. We conclude that this fluorescent analog of tau is associated with microtubules, the nucleolus, and other microtubule‐related structures in living cells, and is competent to stabilize microtubules against microtubule depolymerizing drug treatment. This approach provides a useful model system for the study of modified tau in neurodegenerative disease. © 1993 Wiley‐Liss, Inc.
Intracellular guanosine‐5′‐0‐(3‐thiotriphosphate) blocks chemotactic motility of Dictyostelium discoideum amoebae
Authors: Schlatterer Christina , Malchow Dieter 
Published: February 2005

AbstractStarving amoebae of the cellular slime mold Dictyostelium discoideum react chemotactically towards the attractant cAMP. In this study, the effect of nonhydrolyzable analogs of GTP and GDP on the chemotactic behavior was analyzed with light microscopic techniques. Guanosine‐5′‐0‐(2‐thiotriphosphate) (GTPβS) or guanosine‐5′‐0‐(2‐thiodiphosphate) (GDPβS) was scrape‐loaded into the cytoplasm of cells, together with a fluorescent marker. Stimulation with a cAMP‐filled glass capillary revealed a reduced capacity of loaded cells to migrate to wards the capillary tip. Most cells still protruded filopods in the direction of the capillary tip, but full extension of pseudopods was inhibited in a dose‐dependent and reversible manner. This indicates that in the presence of the analogs, chemotactic sensing still occurs, and that a more distal step of the cascade of events leading to the formation of the pseudopod is impaired.In cells loaded with the analogs together with the calcium indicator fura‐2, stimulation with 10 μM cAMP led to a transient change in the intracellular free calcium concentration ([Ca2+]i), which was detectable in 28% of the cells. Furthermore, large vacuoles were found containing high amounts of calcium. On the other hand, clamping of [Ca2+]i at low levels with 1,2‐bis(2‐aminophenoxy) ethane N,N,N′,N′‐tetraacetic acid (BAPTA) also inhibited motility, with neither filopods nor pseudopods formed.The data suggest that chemotactic migratory activity involves GTP‐dependent processes that participate in the regulation of the Ca2+ homeostasis of the cell and in the regulation of membrane traffic that contributes to the directed locomotion. © 1993 Wiley‐Liss, Inc.
Localization of CapZ during myofibrillogenesis in cultured chicken muscle
Authors: Schafer Dorothy A. , Waddle James A. , Cooper John A. 
Published: February 2005

AbstractActin filaments undergo dramatic changes in their organization during myofibrilloenesis. In mature skeletal muscle, both CapZ and the barbed end of the actin filaments are located at Z‐discs. In vitro, CapZ binds the barbed end of actin filaments and prevents actin subunit addition and loss; CapZ also nucleates actin polymerization in vitro. Taken together, these properties suggest that CapZ may function to organize actin filaments during myofibrillogenesis. We report here that the amount of CapZ in myofibrils from adult chicken pectoral muscle is sufficient to “cap” each actin filament of the sacromere. Double inmmunofluorescence microscopy of skeletal muscle cells in culture was used to determine the spatial and temporal distributions of CapZ relative to actin, α‐actinin, titin, and myosin during myofibrilloenesis. Of particular interest was the assembly of CapZ at nascent Z‐discs in relation to the organization of actin filaments in nascent myofibrils. In myoblasts and young myotubes, CapZ was diffusely distributed in the cytoplasm. As myotubes matured, CapZ was initially observed in a uniform distribution along non‐striated actin filaments called stress fiber‐like structures (SFLS). CapZ was observed in a periodic pattern characteristic of mature Z‐discs along the SFLS prior to the appearance of a striated staining pattern for actin. In older myotubes, when actin was observed in a pattern characteristic of I‐bands, CapZ was distributed in a periodic pattern characteristic of mature Z‐discs. The finding that CapZ was assembled at nascent Z‐discs before actin was observed in a striated pattern is consistent with the hypothesis that CapZ directs the location and polarity of actin filaments during I‐band formation in skeletal muscle cells. The assembly of CapZ at nascent Z‐disc structures also was observed relative to the assembly of sarcomeric α‐actinin, titin, and thick filaments. Titin and myosin were observed in structures having the organization of mature sarcomeres prior to the appearance of CapZ at nascent Z‐discs. The distribution of CapZ and sarcomeric α‐actinin in young myotubes was not coincident; in older myotubes, both CapZ and α‐actinin were co‐localized at Z‐discs. In cardiac myocytes, CapZ was detected at Z‐discs and was distributed in a punctate pattern throughout the cytoplasm. CapZ also was co‐localized with A‐CAM and vinculin at cell‐cell junctions formed by the myocytes. © 1993 Wiley‐Liss, Inc.
Near‐UV radiation disrupts filamentous actin in lens epithelial cells
Authors: Rafferty Nancy S. , Zigman Seymour , McDaniel Thurma , Scholz Diane L. 
Published: February 2005

AbstractUltraviolet radiation in the near range (UVA) causes lens opacification and disrupts the actin cytoskeleton in rabbit and gray squirrel lenses. Changes were noted using transmission electron microscopy of tangential sections and rhodaminephalloidin fluorescence microscopy of epithelial whole mounts of irradiated and unirradiated lenses, and corresponded with gross cataract formation. Irradiated lenses lacked microfilament polygonal arrays at the inner surface of the apical plasma membrane (i.e., in the cell pole next to the lens fibers) in lens epithelia of both species; a condensed actin bundle was present instead. This bundle, and scattered small actin clumps in the cytoplasm, were identified by immunogold TEM, using a specific antibody and a secondary antibody conjugated with coloidal gold. Similar techniques showed breakdown of tubulin and vimentin, but after longer intervals than for the breakdown of actin. Generalized cytologic damage was also present in epithelial cells, but not in the underlying cortical lens fibers. Damage began to occur after 4 hr of irradiation and became more severe with increased exposure. Shielded controls remained clear, had normal cytology and polygonal arrays, and no clumping of actin filaments. © 1993 Wiley‐Liss, Inc.
Decreased levels of the high molecular weight subunit of neurofilaments and accelerated neurofilament transport during the recovery phase of 2,5‐hexanedione exposure
Authors: Pyle Sally J. , Amarnath Venkataraman , Graham Doyle G. , Anthony Douglas C. 
Published: February 2005

AbstractThe neurotoxicant 2,5‐hexanedione (HD) causes the accumulation of neurofilaments in the distal axon and an acceleration of neurofilament transport proximal to the site of their accumulation. It has been proposed that the acceleration of transport is due to the direct reaction of HD with neurofilament proteins and, conversely, that this acceleration is a secondary response of the axon to injury. The objective of this study was to determine whether the response of axons to HD intoxication includes acceleration of neurofilament transport. Pulse labeling was used to analyze neurofilament transport in age‐matched rats exposed to HD or PBS. The animals receiving HD were exposed either throughout the period of radiolabel transport, or prior to the pulse labeling of neurofilament proteins. If acceleration of the rate of neurofilament transport was due to the direct reaction of HD with proteins, then neurofilaments synthesized after the exposure period should travel at control rates, since these proteins would not have been exposed to the toxicant. After 28 days of transport, optic nerve proteins were examined using SDS‐PAGE, fluorography, and computerized densitometry. In both HD‐treated groups, neurofilament transport was accelerated relative to age‐matched control animals. In addition, the amount of NFH was decreased relative to other neurofilament subunits. The combination of accelerated transport and a diminished proportion of NFH is similar to the observations of neurofilament axonal transport during growth and development. These observations suggest that this persistent, secondary effect is a reparative response to injury that recapitulates axonal growth and development. © 1993 Wiley‐Liss, Inc.
Actin, α‐actinin, and vinculin are associated with septate junctions in insecta
Authors: Colombo Anita , Bonfanti Patrizia , Camatini Marina 
Published: February 2005

AbstractCytoskeletal elements associated with the smooth septate junctions linking the midgut columnar cells of Manduca sexta larvae (Insecta, Lepidoptera) were characterized. Myosin subfragment 1 decoration and immunostaining for actin demonstrated that the filaments associated with the septate junctions were constituted of actin. Moreover, using a combination of immunochemical and immunolocalization techniques, evidence is presented that α‐actinin, myosin II, and vinculin are localized close to the specialized plasma membranes. The insertion of microfilament bundles into submembranous F‐actin/α‐actinin/vinculin complexes, previously described in vertebrate junctions of adherens type, appears to be a more general organization, including the insect septate junction here examined. © 1993 Wiley‐Liss, Inc.
Smooth muscle myosin subfragment‐1 is a kinetic analogue for heavy meromyosin in the extended conformation
Authors: Drew Jean S. , White Marianne P. , Stein Leonard A. 
Published: February 2005

AbstractThe 10S→6S (Flexed→Extended) transition in smooth muscle myosin is related to increased ATPase activity, but there is controversy over whether the analogous 9S→7S transition in HMM is also associated with ATPase activity. We therefore studied the association of ionic strength, phosphorylation, and ATPase activity for HMM as compared to S1 which has no apparent flexed conformation. In addition, we performed both steady state and single turnover analyses, to control for artifacts due to multiple subfragment populations that might skew steady state results.At low ionic strength where myosin and HMM are in the flexed conformation, HMM had a near zero ATPase activity while S‐1 had a high ATPase rate (0.07 s−1). At 400 mM ionic strength, where both myosin and HMM are in the extended conformation, S1 and HMM had the same ATPase rate (0.04 s−1). Phosphorylation did not affect S1 significantly, but shifted the HMM curve to higher rates at lower ionic strengths. Both steady state and single turnover experiments gave the same results, indicating that steady state results were not skewed by multiple subfragment populations. These data indicate that HMM has a conformation‐ATPase relation similar to that observed with myosin. Furthermore, these findings suggest that the S1 ATPase rate corresponds to that of HMM in the extended conformation. © 1993 Wiley‐Liss, Inc.
Differential distribution of glutamylated tubulin during spermatogenesis in mammalian testis
Authors: Fouquet Jean‐Pierre , Kann Marie‐Louise , Edde Bernard , Wolff Annie , Desbruyeres Elisabeth , Denoulet Philippe 
Published: February 2005

AbstractThe distribution of glutamylated tubulin has been analyzed in mammalian testis using the specific mAb GT335 by immunoelectron microscopy and immunoblotting. In spermatozoa of various species, immunogold labeling showed the presence of glutamylated tubulin in all of the microtubules of axoneme and centrioles, whereas the microtubule network of the spermatid manchette was unlabeled. In earlier germ cells, centriole was the only microtubule structure to be labeled. A similar distribution was observed using the anti‐acetylated tubulin antibody (6‐11B‐1), confirming previous results of Hermo et al. [Anat. Rec. 229:31‐50, 1991]. However, among testicular somatic cells, microtubules of some Sertoli cell branches were not acetylated but glutamylated. 2‐D PAGE of mouse and hamster sperm extracts showed a high level of α and β‐tubulin heterogeneity, comparable to that found in brain. Immunoblotting with GT335 revealed a large amount of glutamylated tubulin resolved into numerous α as well as β‐tubulin isoforms. This suggests that the major testis‐specific tubulin isotypes (mα3/7 and mβ3) are also glutamylatable. These results show a subcellular sorting of posttranslationally modified tubulin isoforms in spermatids, glutamylation being associated with the most stable microtubule structures. © 1994 Wiley‐Liss, Inc.
Role of microtubules in random cell migration: Stabilization of cell polarity
Authors: Glasgow James E. , Daniele Ronald P. 
Published: February 2005

AbstractThe role of microtubules in random cell migration was investigated using time‐lapse videomicroscopy to record in vitro the shape and motile behavior of guinea pig alveolar macrophages before and after disrupting microtubules with colcemid. Cell migration was quantified in terms of directional persistence time and speed. Motility was also correlated with morphological polarity: cells having a single lamellipodal region (monopolar cells) migrated, whereas those lacking a lamellipod (apolar cells) or with opposing lamellipodal regions (bipolar cells) did not migrate. Within 2 hours, colcemid caused a shift in polarity from 80% monopolar cells to 40% monopolar and 40% bipolar cells and a corresponding decrease from 80% to 40% in the fraction of migrating cells. Mean persistence time and speed decreased only slightly (approximately 20%) for those cells (still monopolar) which continued to migrate in the presence of colcemid. Persistence time and speed actually increased for many individual cells, indicating that random migration did not require intact microtubules. We conclude that colcemid treatment destabilizes monopolarity, leading to the gradual loss of monopolarity and consequent inhibition of migration. While a cell remains monopolar, it will continue to migrate even in the absence of intact microtubules, but microtubules are required for the long‐term maintenance of cellular monopolarity and, thus, for continued motility. © 1994 Wiley‐Liss, Inc.
Cellular microtubules heterogeneous in their content of microtubule‐associated protein 4 (MAP4)
Authors: Chapin Steven J. , Bulinski Jeannette Chloë 
Published: February 2005

AbstractPrevious immunolocalization studies using many primate cultured cell lines demonstrated that a microtubule‐associated protein of Mr ∼210,000 which is now called MAP4, is present along the length of microtubules in interphase and mitotic cells [Bulinski and Borisy (1980) J. Cell Biol. 87:802–808; DeBrabander et al. (1981) J. Cell Biol. 91:438–455]. Since MAP4 has been implicated as a microtubule stabilizer, we asked whether all classes of microtubules possess an equal complement of MAP4. We have reexamined the cellular distribution of MAP4, using both conventional double‐label immunofluorescence and an antibody blocking technique [Schulze and Kirschner (1987) J. Cell Biol. 104:277–288] to highlight microtubules lacking, or depleted in, MAP4. These techniques have revealed that thin processes extending from monkey kidney cells (TC‐7), and those made by human neuroblastoma cells (IMR‐32) in response to retinoic acid, are often deficient in MAP4 immunoreactivity. Since both types of cellular processes contain stable microtubules, which are enriched in detyrosinated (Glu) tubulin, we tested the ability of MAP4 to bind to microtubules made from pure Glu and pure tyrosinated (Tyr) tubulin in vitro. MAP4 bound to both types of microtubules, and the similar saturation level of MAP4 binding to Glu and Tyr microtubules suggested that differential binding to these forms of tubulin does not contribute directly to a mechanism for segregation of MAP4 on microtubules in vivo. In TC‐7 cells, we also observed MAP4‐depletion on single microtubules, distal regions of broad cytoplasmic extensions, and midbodies of dividing cells. MAP4 depletion may reflect recent, rapid growth of microtubules to which MAP4 has not yet bound, or the presence of other MAPs that may compete with MAP4 for binding sites on the MT. We suggest that different levels of MAP4 on microtubules may directly modulate microtubule dynamics within single cells, as well as other microtubule functions such as those involving microtubule motor activity. © 1994 Wiley‐Liss, Inc.
Axonemes paralyzed by the presence of dyneins unable to use ribose‐modified ATP
Authors: Lark Ellen , Omoto Charlotte K. 
Published: February 2005

AbstractSubstrate analogs are useful for studying the structures of active sites and for distinguishing between similar enzyme activities. Fluorescent ribose‐modified ATP analogs were used to investigate the functional differences between dynein ATPases. These analogs reactivate (support the movement of) sea urchin sperm axonemes, yet they do not reactivate wild‐type Chalmydomonas axonemes. Surprisingly, the analogs reactivate the axonemes of mutants completely missing the outer arm dyneins. Competition experiments using ATP and these analogs provide strong evidence that the analogs bind to all dynein active sites but fail to release a subset of dyneins from rigor. We suggest that this subset of Chlamydomonas outer arm dyneins unable to use the analogs remains in rigor in the presence of the analogs and paralyzes the axoneme. © 1994 Wiley‐Liss, Inc.
Different temporal patterns of expression result in the same type, amount, and distribution of filamin (ABP) in cardiac and skeletal myofibrils
Authors: Price Maureen G. , Caprette David R. , Gomer Richard H. 
Published: February 2005

AbstractThe morphogenesis of functional myofibrils in chick skeletal and cardiac muscle occurs in greatly different time spans, in about 7 and 2 days, respectively. In chick skeletal myogenic cells, one isoform of the 250 kD actin‐binding protein (ABP) filamin is associated with stress fiber‐like structures of myoblasts and early myotubes, then disappears for approximately 4 days, whereupon a second filamin isoform reappears at the Z‐disc periphery. We sought to determine if cardiac myogenesis involves this sequence of appearance, disappearance, and reappearance of a new filamin isoform in a compressed time scale. It was known that in mature heart, filamin is localized at the Z‐disc periphery as in mature (fast) skeletal muscle, and is also associated with intercalated discs. We find that myocardial filamin has an apparent molecular weight similar to that of adult skeletal muscle filamin and lower than that of smooth muscle filamin, and that both skeletal and cardiac muscle contain roughly 200 filamin monomers per sarcomere. Two‐dimensional peptide mapping shows that myocardial filamin is very similar to skeletal muscle filamin. Myocardial, slow skeletal, and fast skeletal muscle filamins are all phosphorylated, as previously shown for filamin of non‐striated muscle. Using immunofluorescence, we found that filamin could not be detected in the developing heart until the 14‐somite stage, when functional myofibrils exist and the heart has been beating for 3 to 4 hours. We conclude that in cardiac and skeletal myogenesis, different sequences of filamin gene expression result in myofibrils with similar filamin distributions and isoforms. © 1994 Wiley‐Liss, Inc.
Physical model of axonemal splitting
Authors: Holwill Michael E. J. , Satir Peter 
Published: February 2005

AbstractA physical model developed to explain microtubule sliding patterns in the trypsintreated ciliary axoneme has been extended to investigate the generation of bending moments by microtubules sliding in an axoneme in which the dublets are anchored at one end. With sliding restricted, a bending moment is developed by the polarized shearing interaction between neighbouring doublets, effected by the activity of dynein arms on doublet N pushing N + 1 in a tipward ( + ) direction. In arrested axonemes in which arms on several contiguous doublets are active, the bending moment causes splitting of the 9 + 2 microtubule array into two or more sets of doublets. In the absence of special constraints, splitting depends only on breaking the circumferential interdoublet links most distorted by the bending moment. The analysis, which permits assignment of arm activity to specific microtubules in each of the observed patterns of splitting, indicates that the axoneme will split between doublet N and N + 1 if arms on doublet N are inactive and arms on either N + 1 or N−1 are active. To produce the observed major splits, dynein arms on the microtubules of roughly one‐half of the axoneme are predicted to be active, in a manner consistent with the switch‐point hypothesis of ciliary motion. Electron microscopic examination indicates that virtually every set of doublets in the split axonemes retains its cylindrical form. Maintenance of cylindrical symmetry can be ascribed to the mechanical properties of the unbroken links, which may resist both tensile and compressive stress, and to active dynein arms. © 1994 Wiley‐Liss, Inc.
Localization of tau and other proteins of isolated marginal bands
Authors: Sanchez Ivelisse , Cohen William D. 
Published: February 2005

AbstractTo determine which proteins were associated with and intrinsic to the marginal band (MB) of microtubules (MTs), we studied protein components of MBs isolated from nucleated erythrocytes by differential detergent solubilization of the membrane skeleton (MS). MBs isolated from dogfish erythrocytes contained major proteins in the tubulin Mr range. A high molecular weight protein of ∼290 kD that bound antibody to syncolin and to heat‐stable brain MAPs was present in the whole cytoskeleton. However, most of it was solubilized by the MB isolation medium, together with the MS. Dogfish erythrocyte cytoskeletons and isolated MBs were examined with polyclonal and monoclonal antibodies against mammalian brain tau and chicken erythrocyte tau. As shown by immunofluorescence and immunoblotting, these antibodies bound to proteins in the 50 to 67 kD range, located along the length of isolated MBs. Two‐dimensional SDS‐PAGE revealed isolated MB proteins of pI ∼6.8 in the same molecular weight range, as well as α‐ and β‐tubulin with pI ∼5.4. Subtilisin or high‐salt treatment of isolated MBs resulted in unbundling of MTs, indicating involvement of MAPs. MBs isolated from chicken erythrocyte cytoskeletons also contained tau as shown by antimammalian brain tau immunofluorescence. Both chicken and dogfish isolated MBs also bound phalloidin, but the binding was usually discontinuous and, for any given MB, matched the pattern of anti‐syncolin binding. Both syncolin and F‐actin were part of the MS remnant remaining after MT disassembly, supporting their assignment to a specialized MS region at the MB/MS interface. In contrast, tau protein appears to be intrinsic to the MB, where it may have an MT stabilizing and bundling function. © 1994 Wiley‐Liss, Inc.