bluuebunny/crossref_metadata_2025_split huggingface dataset:
Decoration of microtubules by fluorescently labeled microtubule‐associated protein 2 (MAP2) does not interfere with their spatial organization and progress through mitosis in living fibroblasts
Authors: Vandenbunder Bernard , Borisy Gary G. 
Published: February 2005

AbstractMicrotubule‐associated protein 2 (MAP2) derivatized with iodoacetamidotetramethylrhodamine or with iodoacetamidofluorescein binds to microtubules after injection into living interphase cells [Scherson et al, 1984]. The binding of derivatized MAP2 stabilized microtubules in vitro; it was therefore important to check if the binding of MAP2 in vivo perturbed the dynamics and organization of the microtubule network. We have addressed these questions by studying the effect of the injection of derivatized MAP2 on mitosis in PtK 1 cells and on the recovery of the microtubule network from low temperature incubation in interphase cells. We found that the presence of derivatized MAP2 did not change the duration of any mitotic stage and that the injected cell normally completed mitosis. We subsequently showed that the injected MAP2 bound to the microtubules within 5 minutes after injection and remained bound throughout the course of mitosis. The reorganization of the microtubule network upon cooling and rewarming was studied in the cytoplasm of human foreskin fibroblasts (356 cells). During the recovery, the distribution of the fluorescent MAP2 in living cells was identical with the microtubule pattern visualized by immunofluorescence in lysed and fixed cells.In these experiments, the fluorescent MAP2 bound to microtubules can be considered as a nonperturbing reporter of the microtubule network. This result is discussed in terms of the role of MAPs in the dynamics and organization of microtubules in living cells.
Cyclical bending of two outer‐doublet microtubules in frayed axonemes of Chlamydomonas
Authors: Kamiya Ritsu , Okagaki Tsuyoshi 
Published: February 2005

AbstractWhen detergent‐extracted cell models of Chlamydomonas reinhardtii were left in the presence of 1 mM Mg‐ATP for more than 30 minutes flagellar axonemes tended to become frayed into fine bundles of microtubules. Under such conditions, bundles made up of a pair of outer‐doublet microtubules displayed oscillatory bending movements of low (< 2 Hz) frequencies. The two doublet microtubules underwent association‐dissociation cycles coupled with gross bending movement. A model is presented to explain this phenomenon by unidirectional sliding interaction between the two microtubules.
Evidence for functional differences between two flagellar dynein ATPases
Authors: Penningroth Stephen M. , Peterson Dolores D. 
Published: February 2005

AbstractEnergy coupling in flagellar motility was investigated using demembranated, reactivated sea urchin spermatozoa (Arbacia punctulata). The ATP‐dependence of ATPase activity was investigated for ATP concentrations ranging from 4 μM to 600 μM ATP. Using Eadie‐Scatchard plot analysis, we identified two axonemal dynein ATPase activities. Their apparent Michaelis constants were calculated to be equal to 4 μM and 161 μM ATP, and they were referred to, respectively, as the high‐affinity dynein ATPase (HADA) and the low‐affinity dynein ATPase (LADA). Investigation of movement‐coupled ATPase activity (difference between the ATPase activities of reactivated and broken, immotile spermatozoa) indicated that HADA and LADA were both 65% movement‐coupled. The apparent Michaelis constants of movement‐coupled HADA and LADA, 12 μM and 271 μM ATP, respectively, were two‐ to four‐fold greater than the apparent Michaelis constants of movement‐uncoupled HADA and LADA. The apparent Michaelis constants for force generation and beat frequency of reactivated spermatozoa were determined to be 24 μM and 290 μM ATP, respectively. These results raise the possibility that flagellar force generation is controlled primarily by movement‐coupled HADA, and that flagellar beat frequency is controlled primarily by movement‐coupled LADA. Thus, mechanochemical activity in flagellar motility may be divided between two enzymatically and functionally distinct classes of flagellar dyneins.
Characterization of alpha‐actinin from Acanthamoeba
Authors: Pollard Thomas D. , Tseng Peter C.‐H. , Rimm David L. , Bichell David P. , Williams Robley C. , Sinard John , Sato Masahiko 
Published: February 2005

AbstractCharacterization of a protein from Acanthamoeba that was originally called gelation protein [T.D. Pollard, J. Biol. Chem. 256:7666–7670, 1981] has shown that it resembles the actin filament cross‐linking protein, alpha‐actinin, found in other cells. It comprises about 1.5% of the total amoeba protein and can be purified by chromatography with a yield of 13%. The native protein has a molecular weight of 180,000 and consists of two polypeptides of 90,000 Da. The Stokes' radius is 8.5 nm, the intrinsic viscosity is 0.35 dl/dm, and the extinction coefficient at 280 nm is 1.8 × 105M−1·cm−1. Electron micrographs of shadowed specimens show that the molecule is a rod 48 nm long and 7 nm wide with globular domains at both ends and in the middle of the shaft. On gel electrophoresis in sodium dodecylsulfate the pure protein can run as bands with apparent molecular weights of 60,000, 90,000, 95,000, or 134,000 depending on the method of sample preparation. Rabbit antibodies to electrophoretically purified Acanthamoeba alpha‐actinin polypeptides react with all of these electrophoretic variants in samples of purified protein and cell extracts. By indirect fluorescent antibody staining of fixed amoebas, alpha‐actinin is distributed throughout the cytoplasmic matrix and concentrated in the hyaline cytoplasm of the cortex. The protein cross‐links actin filaments in the presence and absence of Ca++. It inhibits slightly the time course of the spontaneous polymerization of actin monomers but has no effect on the critical concentration for actin polymerization even though it increases the apparent rate of elongation to a small extent. Like some other cross‐linking proteins, amoeba alpha‐actinin inhibits the actin‐activated ATPase of muscle myosin subfragment‐1. Although Acanthamoeba alpha‐actinin resembles the alpha‐actinin from other cells in shape and ability to cross‐link actin filaments, antibodies to amoeba and smooth muscle alpha‐actinins do not cross react and there are substantial differences in the amino acid compositions and molecular dimensions.
Effect of microinjectecd calcium‐calmodulin on mitosis in PtK2 cells
Authors: Keith Charles H. 
Published: February 2005

AbstractCalcium and calmodulin are believed in play a significant role in the regulation of mitosis, because they are both localized in the mitotic spindle and because they can potentiate microtubule depolymerization in the test tube and in the living cell. It has been hypothesized, specifically, that calcium‐saturated calmodulin drives the shortening of the kinetochore microtubules that must occur during prometaphase, when the chromosomes congress to the metaphase plate, and during anaphase A, when the half‐spindles shorten. We have examined the role of calmodulin in mitosis by observing the consequences of calmodulin microinjection on the progress of mitosis and morphology of the mitotic spindle in PtK2 cells. We have found that the injection of excess calcium‐saturated calmodulin during early prometaphase significantly prolongs the time required for the cell to go into anaphase, and that neither calcium‐depleted calmodulin nor buffer alone produce a similar perturbation. Calcium ion alone produces a similar but much smaller retardation of mitosis. Immunofluorescence and fluorescent analogue cytochemical studies of spindle morphology reveal that the immediate (<5‐min) effect of calcium‐saturated calmodulin on prometaphase spindles is a significant shortening of the kinetochore fibers and “interpolar” microtubules but not the astral microtubules. After this perturbation, however, the spindle quickly recovers its normal form. An equivalent transient shortening of the spindle fibers is seen following the injection of calcium chloride solutions but not after the injection of calciumdepleted calmodulin or buffer alone. Taken together, these observations suggest that calcium‐saturated calmodulin plays a significant role in the regulation of mitosis, and that this regulatory pathway involves more than spindle fiber shortening.
Caenorhabditis elegans spermatozoa assemble membrane proteins onto the surface at the tips of pseudopodial projections
Authors: Pavalko Fredrick M. , Roberts Thomas M. 
Published: February 2005

AbstractThe crawling movement of nematode sperm, like that of many other crawling metazoan cells, is accompanied by movement of membrane components from the leading edge of the cell rearward. We used colloidal gold conjugates of monoclonal antibodies (CGP‐ABY) to membrane proteins on Caenorhabditis elegans sperm to examine this surface movement by electron microscopy. Antibody binding sites on fixed sperm are distributed uniformly over the cell surface. However, blocking these sites on live sperm with unlabelled antibody or removing them with protease and then pulse‐labelling the cell with CGP‐ABY revealed that new antigen is assembled onto the surface at the tips of the stubby projections that stud the pseudopod surface. These proteins then move rearward rapidly so that the pseudopod surface pool of antigen is replaced within 2 min. The same pattern of surface movement was observed when live cells were labelled with CGP‐ABY and then washed with buffer before fixation. Bound CGP‐ABY was cleared first from the tips of the projections and subsequently from the entire pseudopod surface. These gold particles accumulated at the base of the pseudopod without moving onto the cell body or being internalized. We did, however, detect a pool of antigen in the pseudopod cytoplasm that may be available for assembly onto the pseudopod surface. We propose that the localized assembly of new membrane and its subsequent rearward movement may play an important role in sperm locomotion.
Stress fiber and cleavage furrow formation in living cells microinjected with fluorescently labeled α‐actinin
Authors: Sanger Jean M. , Mittal Balraj , Pochapin Mark B. , Sanger Joseph W. 
Published: February 2005

Abstractα‐Actinins, isolated from muscle and nonmuscle sources and labeled with various fluorescent dyes, were microinjected into living PtK2 cells during interphase to observe the reformation of stress fibers following cell division. Fluorescently labeled ovalbumin and bovine serum albumin were also injected as control proteins. α‐Actinin was incorporated into stress fibers within 5 minutes after injection and remained present in the fibers for up to 11 days. The pattern of incorporation was the same regardless of whether the α‐actinin was isolated from muscle or nonmuscle tissues or whether it was labeled with fluorescein, Lucifer Yellow, or rhodamine dyes. In contrast, neither labeled ovalbumin nor bovine serum albumin were incorporated into stress fibers. When the injected cells entered prophase, all stress fibers disassembled, resulting in a distribution of the fluorescent α‐actinin throughout the cytoplasm. During cytokinesis, the fluorescent α‐actinin was concentrated in the broad area between the separated chromosomes and along the edge of the cell in the cleavage area. Within 10 minutes after the completion of cleavage, the first fluorescent stress fibers reformed parallel to the spreading edges of the daughter cells and in close association with the midbody with a concomitant loss of α‐actinin in the former cleavage furrow. Additional fibers formed adjacent to these first stress fibers. In some cases, new stress fibers formed between two existing stress fibers and some stress fibers moved up to 4 μm apart from one another in the course of 2 hours. Thus, fluorescent α‐actinin, injected into living cells, undergoes the same cyclical changes in distribution as endogenous α‐actinin during the cell cycle: from stress fibers to cleavage furrow and back to stress fibers.
Evidence that MAP‐2 may be involved in pigment granule transport in squirrel fish erythrophores
Authors: Stearns Mark E. , Binder Lester I. 
Published: February 2005

AbstractWe have demonstrated the presence of MAP‐2 in squirrel fish erythrophores using SDS‐PAGE, immunobolt, and immunoprecipitation techniques. The monoclonal antibodies used (AP‐9, ‐13, ‐14) were raised against distinct antigenic sites on Chinese hamster brain MAP‐2. Immunoprecipitation studies demonstrated that all three antibodies bind a 300 K protein found in crude cell extracts and in partially purified MAP fractions isolated from erythrophores of the squirrel fish Holocentrus rufus. Immunofluorescent studies confirmed that the 300 K protein was present in cultured erythrophores. Studies of cells induced to aggregate and disperse their pigment granules revealed that the 300 K protein comigrated with the pigment, suggesting that the 300 K protein may constitute part of the “α‐cytomatrix” involved in pigment translocations.
Mitotic inhibition and chromosome displacement induced by estradiol in chinese hamster cells
Authors: Wheeler W. J. , Hsu T. C. , Tousson A. , Brinkley B. R. 
Published: February 2005

AbstractWe tested diethylstilbestrol (DES) and 17 β‐estradiol as mitotic arrestants to determine their effects on chromosome distribution, spindle microtubules, and the cytoplasmic microtubule complex (CMTC) in the Chinese hamster strain Don. Cytological experiments assessed micronuclei induction, chromosome displacement, and anaphase recovery Indirect immunofluorescence microscopy with antibody to tubulin and electron microscopy were used to illustrate effects on microtubules. Both DES and estradiol were potent inhibitors of mitosis when applied to cells in vitro. Estradiol induced micronuclei at a greater frequency than did DES. Estradiol‐arrested metaphases often contained misaligned chromosomes despite the presence of a bipolar spindle and an equatorial plate. Equatorial plates were not observed in DES‐arrested cells. Cells recovered quickly from estradiol exposure upon removal of the steroid. The frequency of abnormal metaphases and abnormal anaphases declined as the recovery period increased. Microtubule experiments showed that DES inhibited spindle assembly and disassembled the CMTC, whereas estradiol, at similar concentrations, arrested mitosis in a manner that allowed spindle assembly. A definite effect on the CMTC by estradiol could not be determined. However, changes in cell morphology were observed. In the presence of estradiol, centrosomes organized microtubules that joined with kinet‐ochores of chromosomes at the equatorial plate as well as with those of misaligned chromosomes. Misaligned chromosomes appeared predominantly at polar regions of mitotic cells. Following drug removal, the pole‐oriented chromosomes reoriented at the equatorial plate. The unique arresting properties of estradiol may prove useful in studies of chromosome migration and segregation during mitosis.
Cytoskeletal and myonemal structures of dinoflagellates are made of 2–3‐nm filaments
Authors: Cachon J. , Cachon M. , Boillot A. , Brown D. L. 
Published: February 2005

AbstractAn important cortical fibrillar cytoskeleton is described in some unarmoured dinoflagellates (Gymnodinium, Gyrodinium). The filaments which constitute this cortical layer are 2–3 nm in diameter, tightly packed, entangled, and resistant to denaturing agents. Issuing from them are other bundles of filaments which appear to be involved in the contraction of the cell, and for this reason they are termed “myonemal.”In highly contractile unarmoured dinoflagellates (Leptophyllus) the cytoskeleton is also made of 2–3‐nm filaments, but they are regularly arrayed and more directly involved in contraction and thus correspond also to myonemal structures.Both structures—cytoskeletal and myonemal—are made of 2–3‐nm filaments.
3T3 cell motility in the temperature range 33°C to 39°C
Authors: Thurston G. , Palcic B. 
Published: February 2005

AbstractThe phenomena of mammalian cell motility in tissue culture is an integrated function of many cellular components. As such, cell motility is very sensitive to external stimuli and perturbation. In this article we report the effect of temperature in the range 33°C to 39°C on cell motility. For this 3T3 cells were plated in plastic tissue culture flasks. A large number of individual cells (60 per experiment) were tracked as a function of time by means of an automated device, the Cell Analyzer. The data show a peak in the average cell speed in the range 36.5°C to 38.5°C, falling off sharply at lower and higher temperatures. The average rate of cell motility closely correlates to the average cell proliferation rate in the range 33°C to 39°C.
Microtubule cycle in Chlamydomonas reinhardtii: An Immunofluorescence study
Authors: Doonan John H. , Grief Christopher 
Published: February 2005

AbstractMonoclonal antibodies to yeast tubulin have been used to investigate the distribution of microtubules throughout the cell cycle of the green alga Chlamydomonas reinhardtii. By indirect immunofluorescence microscopy we detect all the classes of microtubule‐containing structures previously described from ultrastructural investigations but we can now demonstrate the complex spatial and temporal relationships between the different microtubule arrays. During interphase a cortical cytoplasmic microtubule system emanates from the base of the flagellar microtubules. These microtubules become reorganised on entry into mitosis, being largely disassembled and replaced by the spindle and “metaphase band” microtubules. The “metaphase band” is shown to be not one array but two distinct sets of microtubules, each linking a spindle pole with a region of the cell cortex near the spindle equator. It persists until late telophase, when it is replaced by cortical and cleavage furrow microtubules.
Isolation and initial biochemical characterisation of caps of two major rat thymocyte glycoprotens: Evidence for the involvement of a 205 K Con A binding protein and cytoskeletal components in capping
Authors: Tuner Christopher E. , Shotton David M. 
Published: February 2005

AbstractTwo major rat thymocyte surface glycoproteins, the leucocyte‐common (L‐C) antigen and the leucocyte sialoglycoprotein (LSGP), were induced to cap independently, using the specific monoclonal antibodies OX‐1 and W3/13, respectively, and an appropriate fluorescently labeled second antibody layer. The caps were subsequently isolated from detergent extracted cells by a procedure involving gentle shearing.TRITC‐phalloidin staining of the isolated caps demonstrated the presence of F‐actin within these structures, and lectin‐affinity staining after fractionation on SDS polyacrylamide gels revealed the presence of a concanavalin A (Con A) binding protein of relative molecular weight (Mr) 205,000, gp205, in both the L‐C antigen and LSGP caps, but absent from the detergent‐insoluble residue isolated from unchallenged cells. These results suggest that gp205 may be involved in the association of cross‐linked glycoproteins with the cytoskeleton during capping.
Topographical relationship between the axonemal arrangement and the bend direction in starfish sperm flagella
Authors: Mohri Hideo , Mohri Toshiko , Okuno Makoto 
Published: February 2005

AbstractSince starfish spermatozoa have spherical heads, it is not easy to determine the topographical relationship of the axoneme to the directions of the flagellar bends, the principal, and the reverse bends as defined by Gibbons and Gibbons [J. Cell. Biol. 1972, 63:970–985]. The demembranated spermatozoa are known to take the quiescent “cane” shape with a sharp principal bend at the proximal region of the flagellum in the presence of high concentration of Ca2+. When such spermatozoa were placed on a grid for electron microscopy, fixed with osmic acid vapor, washed with distilled water, and negatively stained with urany1 acetate, the head of the spermatozoon was disrupted and dispersed disclosing the proximal centriole at at the proximal end of the flagellum. The proximal centriole was always found on the concave side of the “cane” ‐shaped flagella. Electron microscopy of the serial thin sections of intact and demembranated spermatozoa revealed that the doublet microtobules numbers 5 and 6 were contained in the convex edge of the principal bend.
Platelet‐derived growth factor‐induced alterations in vinculin distribution in porcine vascular smooth muscle cells
Authors: Herman B. , Roe M. W. , Harris C. , Wray B. , Clemmons D. 
Published: February 2005

AbstractExposure of porcine vascular smooth muscle cells to platelet‐derived growth factor (PDGF; 18–180 ng/ml) but not epidermal growth factor (EGF; 30ng/ml), somatomedin C (SmC; 30 ng/ml), or insulin (10 μM), results in a rapid, reversible, time‐ and concentration‐dependent disapperance of vinculin staining in adhesion plaques; actin‐containing stress fibers also become disrupted following exposure of cells to PDGF. Disapperance of vinculin staining from adhesion plaques is also caused by 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA; 200–400 nM), though the time course of the disapperance of vinculin staining under these conditions takes longer than in cells exposed to PDGF. The PDGF‐induced removal of vinculin from adhesion plaques was inhibited in a concentration‐dependent fashion by 8‐(N, N‐diethylamin) octy1–3,4,5‐trimethoxybenzoate (TMA‐8; 0.25–4 μM) and leupepetin (2–300 μM), and by n‐α‐rosyl‐L‐lysine chloromethylketone (TLCK; 100 μM) and trifluoperazine (TFP; 2.5 μM). Addition of PDGF to vascular smooth muscle cells caused a rapid, tranient increase in cytosolic free calcium, from a basal resting level of 146 ± 6.9 nM (SEM, n=62) to 414 ± 34 nM (SEM, n=22) as determined using the calcium‐sensitive indicator Fura‐2 and Digitized Video Microscopy. This increase in cellular calcium preceded the disappearance of vinculin from adhesion plaques and was partially blocked by pretreatment of cells with TMB‐8 but not leupeption. This rise in cytosolic free calcium was found to occur in ∼ 80% of the sample population and dispalyed both spatial and temporal subcellular heterogeneity. Exposure of cells to TPA (100 nM) did not result in a change in cytosolic free calcium. Both PDGF (20 ng/ml) and TPA (100 nM) caused cytosolic alkalinization which occurred after PDGF‐induced disruption of vinculin from adhesion plaques, as determined using the pH‐sensitive indicator BCECF and Digitized Video Microscopy. PDGF stimulated DNA synthesis and vinculin disruption in a similar dose‐dependent fashion. Both could be inhibited by leupeptin or TMB‐8. These results suggest that 1) exposure of vascular smooth muscle cells to PDGF is associated with the disruption of vinculin from adhesion plaques, 2) PDGF‐induced vinculin disruption is regulated by an increase in cytosolic calcium (but not cytosolic alkalinization), and involves proteolysis; 3) activation of protein kinase C also causes vinculin removal from adhesion plaques but by a calcium‐independent mechanism, and 4) the cellular response to PDGF‐stimulated increases in cytosolic free calcium is heterogeneous. Our data also suggest that cytosolic vinculin distribution is a sensitive indicator of the response of vascular smooth muslce cells to PDGF.