bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution of detyrosinated microtubules in motile NRK fibroblasts is rapidly altered upon cell‐cell contact: Implications for contact inhibition of locomotion
Authors: Nagasaki T. , Chapin C. J. , Gundersen G. G.
Published: February 2005

Fibroblasts migrating into an experimental wound contain an extensive array of detyrosinated microtubules (Glu MTs) oriented in the direction of migration, whereas nonmotile cells in the interior of a monolayer contain Glu MTs that are primarily coiled around the nucleus. To determine the role of cell‐cell contact in the formation of these distinct arrays of Glu MTs, we studied the distribution of Glu MTs by immunofluorescence in NRK fibroblasts that had been fixed at different intervals after they had established contact with other cells. Time‐lapse video recordings were made of the contacting cells to provide a record of cellular behavior. In motile cells that became completely surrounded by virtue of contact with other cells, Glu MTs were found mostly coiled around the nucleus. The proportion of cells whose Glu MTs extended to the original leading edge decreased dramatically after the cells had been surrounded for 10 min or more. At earlier times, when the contact was confined to a portion of the cell margin, Glu MTs were absent from the area behind the contact site, yet were still oriented toward the noncontacting and ruffling margins. The contact‐induced alteration of Glu MTs was not due to the cessation of forward locomotion of cells per se, since immobilization of cells with cytochalasin D did not cause a dramatic change in Glu MTs. That cell‐cell contact also specifies the type of Glu MTs formed in cells was shown by experiments in which MTs were regrown following complete depolymerization with nocodazole. The remodeling of Glu MTs during cell‐cell contact may be involved in cellular repolarization during contact inhibition of locomotion and will be a useful marker for further dissecting the molecular events of contact inhibition of motility. © 1992 Wiley‐Liss, Inc.
A high molecular weight centrosomal protein of mammalian cells is antigenically related to myosin II
Authors: Bailly Eric , Bordes Nicoles , Bornens Michel , Klotz Catherine
Published: February 2005

Available data on the molecular composition of the centrosome, the typical microtubule‐organizing center of animal cells, are still fragmentary. To address this important issue we have taken advantage of centrosome isolation from a human lymphoblastic cell line (KE37) to generate a monoclonal antibody (mAb) library. Here we present the characterization of one of these mAbs (CTR56). On the basis of both its immunofluorescence staining pattern and its reactivity with a major 200 kD antigen on immunoblots, CTR56 has been tentatively classified as an anticellular myosin heavy chain. In light of cytological and biochemical data obtained in parallel with two other well‐characterized myosin antibodies, it appears that myosin cannot be considered as a genuine centrosomal protein. We have resolved the paradoxical results with CTR56 by showing that in addition to the cellular myosin heavy chain, this antibody also recognizes a high molecular weight protein specifically enriched in centrosomal fractions. The possible biological significance of this finding is discussed in structural and functional terms. © 1992 Wiley‐Liss, Inc.
Cap100, a novel phosphatidylinositol 4,5‐ bisphosphate‐ regulated protein that caps actin filaments but does not nucleate actin assembly
Authors: Hofmann Andreas , Eichinger Ludwig , André Elisabeth , Rieger Daniela , Schleicher Michael
Published: February 2005

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

Using several in vitro motility assays, we found that motility driven by the microtubule (MT) motors, kinesin and cytoplasmic dynein, could be inhibited by MAP2 but not by tau protein or the MT‐binding proteolytic fragment of MAP2.

In MT gliding assays, even the presence of one MAP2 molecule per sixty‐nine tubulin dimers caused an inhibition of about 75% of MT motility at low concentrations of both motors. The percent inhibition of motility decreased with increasing concentration of either motor, suggesting that the inhibition was the result of competition for access to the MT surface. The decrease in the number of moving MTs with MAP2 was correlated with an increase in the frequency of release of moving MTs from the motor‐coated glass. In assays of in vitro vesicular organelle motility and formation of ER networks, the presence of MAP2 inhibited small vesicle movements and to a lesser extent ER network formation.

To determine if competition for specific sites on the MT or coating of the MT surface inhibited motility, we used tau protein and the chymotryptic MT‐binding fragments of MAP2 to coat MTs. No inhibition was observed and there was even an increase in the number of attached and moving MTs in the gliding assay with tau‐coated MTs. Because MAP2, tau and the chymotryptic MT‐binding fragments of MAP2 bind to the same domain on tubulin, masking of the MT surface sites does not appear responsible for the inhibition of motility by MAP2. Rather, we suggest that the sidearm of MAP2 interfered with the interaction of motors with MTs and caused a dramatic increase in the rate of MT release. In vivo, MAP2 could play a major role in the generation of cellular polarity even at substoichiometric levels by inhibiting transport on microtubules in specific domains of the cytoplasm. © 1993 Wiley‐Liss, Inc.
Cytoskeletal sheets of mammalian eggs and embryos: A lattice‐like network of intermediate filaments
Authors: Capco David G. , Gallicano G. Ian , McGaughey Robert W. , Downing Kenneth H. , Larabell Carolyn A.
Published: February 2005

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

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

We have developed a method for producing sea urchin egg cytoplasmic extracts which support substantial microtubule‐associated motility, particularly minus end‐directed motility characteristic of cytoplasmic dynein. Particles translocated along microtubules and axonemes predominantly in the minus end direction; microtubules and axonemes glided across the coverslip surface only in the plus end direction (as expected for a minus‐end directed motor bound to the coverslip surface); and microtubules crosslinked into bundles in an antiparallel orientation. Velocities of particle and microtubule translocation were in the range of 0.5–1.8 μm/sec. Vanadate at 10 μM inhibited all gliding of the microtubules and axonemes, yet bidirectional particle transport persisted. Vanadate at concentrations of 25 μM and higher inhibited nearly all microtubule‐based motility in the preparation and produced parallel bundling of the microtubules. Motility was slowed but not stopped in the presence of 5 mM AMP‐PNP.

Usually when a particle bound to a microtubule wall, it moved to the microtubule minus end. These particles often remained attached to the minus end. When a microtubule plus end in the shortening phase of dynamic instability reached a stationary particle on the microtubule, sometimes normal minus enddirected motility was activated, or at other times the particle remained attached to the shortening plus end. © 1993 Wiley‐Liss, Inc.
Microinjection of fluorescent brain tubulin reveals dynamic properties of cortical microtubules in living plant cells
Authors: Wasteneys Geoffrey O. , Gunning Brain E. S. , Hepler Peter K.
Published: February 2005

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

NG108‐15 cells extend “rapid‐onset” neurites vigorously within the first hour after plating in minimal serum‐free medium on Petri dishes coated with polylysine and laminin (1 ng/mm2). We recently reported that the initial rates of neurite formation and cell translocation are further accelerated in this system when nonspecific substratum attachment sites are partially blocked by polyglutamate, bovine serum albumin, or polyethylene glycol polymers [Smalheiser, N. R. (1991): Dev. Brain Res. 62:81–89]. When cells were plated in the presence of the monovalent cation ionophore monensin (1–5 μM) or hypertonic sucrose (50–100 mM), the initial rate of outgrowth on laminin/polylysine‐treated Petri dishes was not affected, yet the acceleration produced by polyglutamate was strongly inhibited. These data indicate that monensin‐sensitive intracellular events can regulate neurite extension on laminin indirectly, through modulating the effects exerted on cells by nonspecific substratum sites. Although the critical events affected by monensin remain to be identified, movements of laminin receptors (their clustering, internalization, and recycling) are likely targets for further study. © 1993 Wiley‐Liss, Inc.
FRAP analysis of the stability of the microtubule population along the neurites of chick sensory neurons
Authors: Edson Kathryn J. , Lim Soo‐Siang , Borisy Gary G. , Letourneau Paul C.
Published: February 2005

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

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

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

This report describes the ontogenesis of cochlear stereocilia using scanning electron microscopy for analysis of cilia appearance, and fluorescence microscopy of phalloidin, a label for F‐actin, to determine the maturation of the cilia framework. Surface and frozen‐sectioned preparations of the otic capsule were obtained from several stages of rat pup development beginning at the 16th gestational day and at various stages until adulthood. In the earliest stage investigated, strong fluorescence labeling was visible on the apical part of Kölliker's organ, revealing a reticular outline of cell junctions. Hair cells started to differentiate at the 18th day of gestation from cells within the primordial receptor area. Phalloidin labeling revealed a sequential appearance of F‐actin as the hair cells differentiated from the cells within the Kölliker's organ. The differentiation of receptor cells occurred first with the appearance of a junctional complex between the hair cell and the surrounding cells. Then a cuticular plate appeared followed by the progressive emergence of stereocilia.

The F‐actin labeling also revealed a progressive differentiation of receptors cells from the cochlear base to its apex. There was also an inner to outer hair cell developmental gradient of label. Inner hair cells developed stereocilia before outer hair cells. The third row of outer hair cells was the last to acquire stereocilia. The adult patern of stereocilia was reached around the 6th postnatal day.

We conclude that the appearance of actin filaments in developing receptor cells and the emergence of stereocilia can be regarded as markers for correlating function and other structural differentiation. © 1993 Wiley‐Liss, Inc.
Motility of intracellular particles in rat fibroblasts is greatly enhanced by phorbol ester and by over‐expression of normal p21N‐ras
Authors: Alexandrova Antonina Y. , Dugina Vera B. , Paterson Hugh , Bershadsky Alexander D. , Vasiliev Jury M.
Published: February 2005

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

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

A large number of cellular functions require assembly of actin and myosin and coordinated interactions between the resulting filaments. To better understand the structure and function of one such contractile assembly, we have begun fractionation and reconstitution studies of Dictyostelium cytoskeletons. Isolated cytoskeletons rapidly contracted when mixed with Mg‐ATP, and myosin II was essential for this since myosin‐depleted (stripped) cytoskeletons failed to contract. Dictyostelium, Acanthamoeba, or skeletal muscle myosins bound to stripped cytoskeletons with equal efficiency, and the Mg‐ATPase of all three myosins was stimulated by the cytoskeleton‐associated actin. Near neutral pH, however, only the homologous system reconstituted with Dictyostelium myosin contracted, despite the fact that under the same conditions all three myosins bound to myosin‐depleted (ghost) muscle myofibrils and restored contractility. Individual Dictyostelium myosin thick filaments have a strong tendency to aggregate and associate end‐to‐end, and this may be important for functional contraction of cytoskeletons. This suggestion is supported by the observation that under conditions where individual Acanthamoeba myosin filaments aggregated, reconstituted cytoskeletons contracted. None of the solution conditions tested caused rabbit muscle myosin filaments to aggregate or to contract cytoskeletons. Thus higher order associations among individual myosin filaments may be essential for some types of cell motility. © 1993 Wiley‐Liss, Inc.
Disorganization of microfilaments and intermediate filaments interferes with the assembly and stability of desmosomes in MDCK epithelial cells
Authors: Pasdar Manijeh , Li Zhi
Published: February 2005

To investigate the possible role(s) of cytoskeletal elements in desmosome assembly we have studied the effects of cytostatic drugs on the assembly of desmosomes in MDCK epithelial cells. We showed previously [Pasdar et al.: Cell Motil. Cytoskeleton 23:201–213, 1992] that selective disruption of microtubules has no effect on desmosome assembly. Here, we have treated MDCK cells with cytochalasin B and a combination of cytochalasin B and nocodazole and analysed the effects on desmosome assembly. Immunofluorescence analysis of MDCK cultures following drug treatment indicated complete disruption of actin microfilaments and disorganization of cytokeratin intermediate filaments. Biochemical analysis of newly synthesized desmosomal membrane core glycoproteins as well as the cell adhesion proteir. E‐cadherin revealed no effect of these drugs on the kinetics of synthesis, intracellular processing, or transport to the plasma membrane either in the presence or absence of cell‐cell contact. However, morphological analyses revealed a significant disruption in the spatial organization of desmosomal proteins and E‐cadherin. Drug treatment in the absence of cell‐cell contact resulted in the disruption of the normally observed homogenous punctate staining pattern and appearance of aggregate staining. Induction of cell‐cell contact in these cultures resulted in redistribution of some of the aggregate staining to the plasma membrane. In contrast to control cultures, significant amount of intracellular staining was retained for all desmosomal proteins. Biochemical analyses of turnover rates of newly synthesized desmosomal proteins indicated a significant decrease in metabolic stability of these proteins while the turnover rate of E‐cadherin was not significantly different among control and drug‐treated cultures. Taken together, these results suggest that intact actin and cytokeratin filaments are necessary for the stability, efficient assembly, and spatial organization of the junctional components at the membrane. The regulatory role of cytokeratins and actin filaments in assembly and stability of desmosomes on the plasma membrane is discussed. © 1993 Wiley‐Liss, Inc.
CENP‐F is a ca 400 kDa kinetochore protein that exhibits a cell‐cycle dependent localization
Authors: Rattner J. B. , Rao A. , Fritzler M. J. , Valencia D. W. , Yen T. J.
Published: February 2005

We have identified a novel .ca 400 kDa cell‐cycle dependent kinetochore associated protein in human cells, designated CENP‐F, using human autoimmune serum. Immunofluorescence staining using the native serum, affinity purified antibodies, or antibodies raised against a cloned portion of CENP‐F first reveals CENP‐F homogeneously distributed throughout the nucleus of HeLa cells in the G2 stage of the cell cycle. Progression into prophase is accompanied by the localization of CENP‐F to all the kinetochore regions of the karyotype. Kinetochore association is maintained throughout metaphase, but at the onset of anaphase CENP‐F is no longer detected in association with the kinetochore but is found at the spindle mid‐zone. By telophase, it is concentrated into a narrow band on either side of the midbody. Studies of the interaction of CENP‐F with the kinetochore indicate that this protein associates with the kinetochore independent of tubulin and dissociation is dependent on events connected with the onset of anaphase. Nuclease digestion studies and immunoelectron‐microscopy indicate that CENP‐F is localized to the kinetochore plates and specifically to the outer surface of the outer kinetochore plate. The distribution of CENP‐F closely parallels that of another high molecular weight kinetochore associated protein, CENP‐E. Comparative studies indicate that there are antibodies in the CENP‐F reactive autoimmune serum that recognize determinants present in the central helical rod domain of CENP‐E. Immune depletion experiments confirm that CENP‐F exhibits the distribution pattern in cells that was seen with the native autoimmune serum. © 1993 Wiley‐Liss, Inc.
Centrosome repositioning immediately following karyokinesis and prior to cytokinesis
Authors: Mack G. , Rattner J. B.
Published: February 2005

The behaviour of the centrosome immediately following cell division in tissue culture cells has been investigated. We find that following karyokinesis, but preceding cytokinesis, sister centrosomes relocate from the spindle poles to a position adjacent to the intercellular bridge. This repositioning is accompanied by the appearance of a microtubule bundle that extends from the poleward region of the cell to the centrosome and increases in length as the centrosome approaches the intercellular bridge. Disruption of this bundle with colcemid interrupts centrosome repositioning. In contrast, centrosome repositioning persists in late mitotic cells grown in the presence of cytochalasin D. However, the position of the microtubule‐centrosome complex within the cell is randomized suggesting that the path, but not the process, of centrosome repositioning is dependent on an intact actin filament network. This study points out, for the first time, that the complex migration of the centrosome preceding mitosis is paralleled by an equally complex set of events following cell division. We suggest that post‐mitotic centrosome repositioning may play a role in ensuring that daughter cells have equal but opposite polarity and may reflect an interrelationship between the establishment of the interphase cytoskeleton and the completion of cytokinesis. © 1993 Wiley‐Liss, Inc.
β‐actin specific monoclonal antibody
Authors: Gimona Mario , Vandekerckhove Joel , Goethals Marc , Herzog Monika , Lando Zeev , Small J. Victor
Published: February 2005

Using a synthetic peptide mimicking the NH2‐terminus of β‐actin we have raised a monoclonal antibody specific for this cytoplasmic actin isoform. Specificity of the antibody was demonstrated by its labelling of the actin polypeptide only in tissues containing the β isoform, by its exclusive recognition of the synthetic β‐actin peptide amongst those mimicking all six vertebrate isoactins, and by its selective recognition of the β‐actin spot in two‐dimensional electrophoresis gels of smooth muscle extracts. The antibody bound to actin filaments in both living and fixed fibroblasts where it labelled the stress fiber bundles and, more predominantly, the peripheral actin rich lamellipodia. The characteristics of the antibody indicate that it should serve as a useful tool for studying isoactin distribution and function. © 1994 Wiley‐Liss, Inc.
Effects of silver ions (Ag+) on contractile ring function and microtubule dynamics during first cleavage in Ilyanassa obsoleta
Authors: Conrad Abigail H. , Stephens Andy P. , Paulsen Avelina Q. , Schwarting Scott S. , Conrad Gary W.
Published: February 2005

The terminal phase of cell division involves tight constriction of the cleavage furrow contractile ring, stabilization/elongation of the intercellular bridge, and final separation of the daughter cells. At first cleavage, the fertilized eggs of the mollusk, Ilyanassa obsoleta, form two contractile rings at right angles to each other in the same cytoplasm that constrict to tight necks and partition the egg into a trefoil shape. The cleavage furrow contractile ring (CF) normally constricts around many midbody microtubules (MTs) and results in cleavage; the polar lobe constriction contractile ring (PLC) normally constricts around very few MTs and subsequently relaxes without cleavage. In the presence of Ag+ ions, the PLC 1) begins MT‐dependent rapid constriction sooner than controls, 2) encircles more MTs than control egg PLCs, 3) elongates much more than control PLCs, and 4) remains tightly constricted and effectively cleaves the polar lobe from the egg. If Ag+‐incubated eggs are returned to normal seawater at trefoil, tubulin fluorescence disappears from the PLC neck and the neck relaxes. If nocodazole, a drug that depolymerizes MTs, is added to Ag+‐incubated eggs during early PLC constriction, the PLC is not stabilized and eventually relaxes. However, if nocodazole is added to Ag+‐incubated eggs at trefoil, tubulin fluorescence disappears from the PLC neck but the neck remains constricted. These results suggest that Ag+ accelerates and gradually stabilizes the PLC constriction by a mechanism that is initially MT‐dependent, but that progressively becomes MT‐independent. © 1994 Wiley‐Liss, Inc.
Microtubule sliding in reduced‐amplitude bending waves of Ciona sperm flagella: Bending waves attenuated by lithium
Authors: Brokaw Charles J.
Published: February 2005

The distinct damped, or attenuated, bending pattern observed when demembranated sperm flagella of the tunicate, Ciona, are reactivated in the presence of 2 mM Li+ has been analysed in detail. In these patterns, bends are initiated at the base of the flagellum, but die out after they start to propagate along the flagellum, so that little or no bending is seen in the distal half of the flagellum. A quantitative descriptive analysis shows that the distinctive feature of this attenuation of bending wave amplitude is an asymmetric interbend decay, or slippage, occuring, on average, only at the transitions between a reverse bend and the preceding principal bend. This attenuation is combined with a significant amount of synchronous sliding in the distal half of the flagellum and a decrease in propagation velocity of transitions between bends in the mid‐region of the flagellum.

Computer simulations demonstrate that the synchronous sliding in the distal half of these flagella can be an entirely passive consequence of the mechanical interaction between active sliding and bending in the basal third of the flagellum and viscous resistances to movement of the distal region of the flagellum through the fluid environment. The current computer models do not contain a mechanism for asymmetric interbend decay that can reproduce these attenuated bending patterns. © 1994 Wiley‐Liss, Inc.
Role of cAMP in the reactivation of demembranated ram spermatozoa
Authors: San Agustin Jovenal T. , Witman George B.
Published: February 2005

Ejaculated ram sperm were demembranated with Triton X‐100, separated from the detergent‐soluble matrix, and reactivated [San Agustin and Witman (1993): Cell Motil. Cytoskeleton 24:264–273]. The percent motility of models prepared from freshly washed sperm was comparable to that of the washed sample before demembranation, regardless of whether cAMP was included in the reactivation medium. However, demembranated models derived from aging or metabolically inhibited sperm exhibited a lower percent reactivation and required cAMP to attain the level of motility of freshly washed sperm. Cyclic AMP was ∼100 times more effective than cGMP. The requirement for cAMP could be bypassed by addition of porcine heart cAMP‐dependent protein kinase (PKA) catalytic subunit to the reactivation medium, demonstrating that cAMP was acting via PKA. The cAMP stimulation of reactivation was not affected by inclusion of the PKA inhibitor PKI(5–24) in the reactivation medium, but was decreased when the models were preincubated with PKI(5–24) prior to reactivation. The cytosol‐free models retained >90% of the sperm PKA activity; therefore, the PKA appears to be anchored to internal sperm structures. This PKA could not be extracted by cAMP or Triton X‐100 alone, but only by cAMP and Triton X‐100 in combination. We conclude that cAMP‐dependent protein phosphorylation is critical for sperm motility, but that the essential protein phosphate sites turn over slowly under our reactivation conditions, so that the cAMP requirement is apparent only in models prepared from sperm having a low internal ATP or cAMP content. Interestingly, reactivation was rapidly blocked by the peptide arg‐lys‐arg‐ala‐arg‐lys‐glu, which has been reported to be a selective inhibitor of cGMP‐dependent protein kinase. © 1994 Wiley‐Liss, Inc.
Microtubule converging centers and reorganization of the interphase cytoskeleton and the mitotic spindle in higher plant Haemanthus
Authors: Smirnova Elena A. , Bajer Andrew S.
Published: February 2005

We analyzed the distribution and orientation of transitory microtubule structures, microtubule converging centers, during interphase and mitosis in endosperm of the higher plant Haemanthus. In interphase the pointed tips of microtubule converging centers are associated with the nuclear envelope. Their orientation gradually reverses during prophase, and the tips tend to point away from the nucleus. From prometaphase through early telophase, microtubule converging centers are present predominantly in the cytoplasm at the polar region. They are either “free” or associated with chromosomes or microtubule bundles. In late telophase, pointed tips of microtubule converging centers are again associated with the reconstructed nuclear envelope and, additionally, they often appear in the phragmoplast area. The orientation of microtubule converging centers seems to be directly correlated to the previously determined microtubule polarity, with the converging tip being minus and the diverging one, plus.

Elevated temperature (35°–37°) enhances the number of microtubule converging centers in the cytoplasm and at the nuclear envelope. This is especially pronounced during the telophase‐interphase transition and in some interphase cells, indicating temperature and stage dependence.

Our data imply that microtubule converging centers bind together MT minus ends and, thus, control the predominant direction of elongation and shortening of microtubule arrays. We argue that these configurations are instrumental during the reorganization of interphase cytoskeleton and mitotic spindle in Haemanthus endosperm. © 1994 Wiley‐Liss, Inc.
Antisense MAP‐2 oligonucleotides induce changes in microtubule assembly and neuritic elongation in pre‐existing neurites of rat cortical neurons
Authors: Sharma Nishi , Kress Yvonne , Shafit‐Zagardo Bridget
Published: February 2005

Microtubule‐associated protein 2 (MAP‐2) is an abundant component of the cytoskeleton present in dendrites and cell bodies of neurons of the CNS. To examine the biological function of MAP‐2, two MAP‐2 antisense (AS) oligonucleotides complementary to the 5′ region of the rat MAP‐2 cDNA were added to rat primary embryonic day 17–18 (E17‐18) cultured cortical neurons 24 h after plating and neurite outgrowth and morphology studied. The treatment of primary cortical cultures with either of the two MAP‐2 AS oligonucleotides resulted in decreased MAP‐2 and reduction in the number of neuritic processes relative to the control or MAP‐2 sense‐treated cultures. By immunostaining and light microscopy the AS‐treated neurons appeared smaller, more rounded, and less intensely stained for MAP‐2 than the untreated or the MAP‐2 sense‐treated cultures. By electron microscopy disorganized microtubules and a reduction in the number of microtubules within neurites of the AS‐treated cultures were observed. We conclude that MAP‐2 continues to be required for microtubule spacing and stability within neurites once they have formed. © 1994 Wiley‐Liss, Inc.
Regulation and evolution of the single alpha‐tubulin gene of the ciliate Tetrahymena thermophila
Authors: McGrath Kathleen E. , Yu Su May , Heruth Daniel P. , Kelly Anne A. , Gorovsky Martin A.
Published: February 2005

The single alpha‐tubulin gene of Tetrahymena thermophila was isolated from a genomic library and shown to encode a single protein. Comparisons of the rates of evolution of this gene with other alpha‐tubulin sequences revealed that it belongs to a group of more evolutionarily constrained alpha‐tubulin proteins in animals, plants, and protozoans versus the group of more rapidly evolving fungal and variant animal alpha‐tubulins. The single alpha‐tubulin of Tetrahymena must be used in a variety of microtubule structures, and we suggest that equivalently conserved alpha‐tubulins in other organisms are evolutionarily constrained because they, too, are multifunctional. Reduced constraints on fungal tubulins are consistent with their simpler microtubule systems. The animal variant alpha‐tubulins may also have diverged because of fewer functional requirements or they could be examples of specialized tubulins. To analyze the role of tubulin gene expression in regulation of the complex microtubule system of Tetrahymena, alpha‐tubulin mRNA amounts were examined in a number of cell states. Message levels increased in growing versus starved cells and also during early stages of conjugation. These changes were correlated with increases in transcription rates. Additionally, alpha‐tubulin mRNA levels oscillate in a cell cycle dependent fashion caused by changes in both transcription and decay rates. Therefore, as in other organisms, Tetrahymena adjusts alpha‐tubulin message amounts via message decay. However the complex control of alpha‐tubulin mRNA during the Tetrahymena life cycle involves regulation of both decay and transcription rates. © 1994 Wiley‐Liss, Inc.
The premyofibril: Evidence for its role in myofibrillogenesis
Authors: Rhee Dukhee , Sanger Jean M. , Sanger Joseph W.
Published: February 2005

When cardiac muscle cells are isolated from embryonic chicks and grow in culture they attach to the substrate as spherical cells with disrupted myofibrils, and over several days in culture, they spread and extend lamellae. Based on antibody localizations of various cytoskeletal proteins within the spreading cardiomyocyte, three types of myofibrils have been identified: 1) fully formed mature myofibrils that are centrally positioned in the cell, 2) premyofibrils that are closest to the cell periphery, and 3) nascent myofibrils located between the premyofibrils and the mature myofibrils. Muscle‐specific myosin is localized in the A‐bands in the mature, contractile myofibrils, and along the nascent myofibrils in a continuous pattern, but it is absent from the premyofibrils. Antibodies to non‐muscle isoforms of myosin IIB react with the premyofibrils at the cell periphery and with the nascent myofibrils, revealing short bands of myosin between closely spaced bands of α‐actinin. In the areas where the nascent myofibrils border on the mature myofibrils, the bands of non‐muscle myosin II reach lengths matching the lengths of the mature A‐bands. With the exception of a small transition zone consisting of one myofibril, or sometimes several sarcomeres, bordering the nascent myofibrils, there is no reaction of these non‐muscle myosin IIB antibodies with the mature myofibrils in spreading myocytes. C‐protein is found only in the mature myofibrils, and its presence there may prevent co‐polymerization of non‐muscle and muscle myosins. Antibodies directed against the non‐muscle myosin isoforms, IIA, do not stain the cardiomyocytes. In contrast to the cardiomyocytes, the fibroblasts in these cultures stain with antibodies to both non‐muscle myosin IIA and IIB. The premyofibrils near the leading edge of the lamellae show no reaction with antibodies to either titin or zeugmatin, whereas the nascent myofibrils and mature myofibrils do. The spacings of the banded α‐actinin staining range from 0.3 to 1.4 μm in the pre‐ and nascent myofibrils and reach full spacings (1.8‐2.5 μm) in the mature myofibrils. Based on these observations, we propose a premyofibril model in which non‐muscle myosin IIB, titin, and zeugmatin play key roles in myofibrillogenesis. This model proposes that pre‐ and nascent myofibrils are composed of minisarcomeres that increase in length, presumably by the concurrent elongation of actin filaments, the loss of the non‐muscle myosin II filaments, the fusion of dense bodies or Z‐bodies to form wide Z‐bands, and the capture and alignment of muscle myosin II filaments to form the full spacings of mature myofibrils. © 1994 Wiley‐Liss, Inc.