bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequential appearance of muscle‐specific proteins in myoblasts as a function of time after cell division: Evidence for a conserved myoblast differentiation program in skeletal muscle
Authors: Lin Zhongxiang , Lu Mei‐Hua , Schultheiss Thomas , Choi John , Holtzer Sybil , Dilullo Camille , Fischman Donald A. , Holtzer Howard
Published: February 2005

Based on the assumption that a conserved differentiation program governs the assembly of sarcomeres in skeletal muscle in a manner analogous to programs for viral capsid assembly, we have defined the temporal and spatial distribution of 10 muscle‐specific proteins in mononucleated myoblasts as a function of the time after terminal cell division. Single cells in mitosis were identified in monolayer cultures of embryonic chicken pectoralis, followed for selected time points (0–24 h postmitosis) by video time‐lapse microscopy, and then fixed for immunofluorescence staining. For convenience, the myoblasts were termed x‐h‐old to define their age relative to their mitotic “birthdate.” All 6 h myoblasts that emerged in a mitogen‐rich medium were desmin+ but only 50% were positive for a α‐actin, troponin‐I, α‐actinin, MyHC, zeugmatin, titin, or nebulin. By 15 h postmitosis, approximately 80% were positive for all of the above proteins. The up‐regulation of these 7 myofibrillar proteins appears to be stochastic, in that many myoblasts were α‐actinin+ or zeugmatin+ but MyHC− or titin− whereas others were troponin‐I+ or MyHC+ but α‐actinin− or α‐actin−. In 15‐h‐old myoblasts, these contractile proteins were organized into nonstriated myofibrils (NSMFs). In contrast to striated myofibrils (SMFs), the NSMFs exhibited variable stoichiometries of the sarcomeric proteins and these were not organized into any consistent pattern. In this phase of maturation, two other changes occurred: (1) the microtubule network was reorganized into parallel bundles, driving the myoblasts into polarized, needle‐shaped cells; and (2) the sarcolemma became fusion‐competent. A transition from NSMFs to SMFs took place between 15 and 24 h (or later) postmitosis and was correlated with the late appearance of myomesin, and particularly, MyBP‐C (C protein). The emergence of one, or a string of ∼ 2 μ long sarcomeres, was invariably characterized by the localization of myomesin and MyBP‐C to their mature positions in the developing A‐bands. The latter group of A‐band proteins may be rate‐limiting in the assembly program. The great majority of rnyoblasts stained positively for desmin and rnyofibrillar proteins prior to, rather than after, fusing to form myotubes. This sequential appearance of muscle‐specific proteins in vitro fully recapitulates myofibrillar assembly steps in rnyoblasts of the myotome and limb bud in vivo, as well as in nonrnuscle cells converted to myoblasts by MyoD. We suggest that this cell‐autonomous myoblast differentiation program may be blocked at different control points in immortalized rnyogenic cell lines. © 1994 Wiley‐Liss, Inc.
Striated fibers in trichomonads: Costa proteins represent a new class of proteins forming striated roots
Authors: Viscogliosi Eric , Brugerolle Guy
Published: February 2005

The production of monoclonal antibodies and the use of biochemical techniques revealed that B‐type costa proteins in trichomonads are composed of several major polypeptides with molecular weight detected between 100 and 135 kDa similar to those found in the A‐type costae. Although differences were observed between the two types in their fine structure, we tested whether proteins composing the two costa types belong to the same protein family. A polyclonal antibody produced against the 118 kDa costa protein of Trichomonas vaginalis also recognized a 118 kDa costa protein in all other trichomonad genera studied so far whether they have A‐ or B‐type costae. Moreover biochemical characteristics of costa proteins indicated that these proteins might represent a novel class of striated root‐forming proteins in addition to centrin, giardin, and assemblin. © 1994 Wiley‐Liss, Inc.
Microfilament‐associated growth cone component depends upon Tau for its intracellular localization
Authors: DiTella M. , Feiguin F. , Morfini G. , Cáceres A.
Published: February 2005

We report here a novel intracellular localization and function of Tau proteins in cultured cerebellar neurons. Immunofluorescence staining of detergent‐extracted cytoskeletons with antibodies specific for Tau proteins revealed intense labeling of growth cone microtubules. Besides, suppression of Tau by antisense oligonucleotide treatment results in the complete disappearance of antigen 13H9, a specific growth cone component with properties of microfilament‐ and microtubule‐associated protein [Goslin et al., 1989: J. Cell Biol. 109:1621–1631], from its normal intracellular location. This phenomenon is unique to neurite‐bearing cells, is not associated with the disappearance of microtubules from growth cones, and is not reversed by taxol, a microtubule‐stabilizing agent. In addition, Tau‐suppressed neurons display a significant reduction in growth cone area and fillopodial number; on the contrary, fillopodial length increases significantly. The alterations in growth cone morphology are accompanied by considerable changes in the phalloidin staining of assembled actin. Taken together, the present results suggest that in developing neurons Tau proteins participate in mediating interactions between elements of the growth cone cytoskeleton important for maintaining the normal structural organization of this neuritic domain. © 1994 Wiley‐Liss, Inc.
Nanometer scale vibration in mutant axonemes of Chlamydomonas
Authors: Yagi Toshiki , Kamimura Shinji , Kamiya Ritsu
Published: February 2005

Flagellar axonemes of sea urchin sperm display high frequency (200–400 Hz) vibration with nanometer scale amplitudes in the presence of ATP [Kamimura and Kamiya, 1992: J. Cell Biol. 116:1443–1454]. To investigate how various axonemal components affect the vibration, we examined vibration in wild‐type and mutant axonemes of Chlamydomonas. At 1 mM ATP, wild‐type axonemes underwent vibration at 100–650 Hz with amplitudes of 4–40 nm. This vibration was similar to, but less regular than, that in sea urchin sperm. Axonemes of the mutants ida1 and ida4 lacking part of the inner arm dynein underwent vibrations indistinguishable from that of wild‐type. The mutant oda1 lacking the entire outer arm underwent vibration at about half the wild‐type frequency. Unexpectedly, the paralyzed mutants pf18 lacking the central pair and pf14 lacking the radial spokes displayed vibration with significantly higher frequencies and smaller amplitudes than those in the wild‐type vibration. These results indicate that the high‐frequency vibration is common to many kinds of mutant axonemes that lack various axonemal substructures, but that its manner is sensitive to the presence of outer arm dynein and the central pair/radial spoke system. Simultaneous measurements of amplitude and frequency in wild‐type and mutant axonemes suggest that the velocity of microtubule sliding in vibrating axonemes is lower than the velocity of sliding under load‐free conditions. The velocity is particularly low in pf18. A possible mechanism is proposed to explain the lower sliding velocity and vibration amplitude in the pf18 axoneme, based on an assumption that central pair/radial spoke system may work to regulate the switching of two antagonizing forces within the axoneme. © 1994 Wiley‐Liss, Inc.
Stimulation of the ATP‐dependent interaction between actin and myosin by a myosin‐binding fragment of smooth muscle caldesmon
Authors: Lin Yuan , Ishikawa Ryoki , Okagaki Tsuyoshi , Ye Li‐Hong , Kohama Kazuhiro
Published: February 2005

We reported previously that smooth muscle caldesmon stimulates the ATP‐de‐pendent interaction between actin and phosphorylated smooth muscle myosin, as monitored by ATPase measurment and in vitro motility assay. Furthermore, this effect changes from stimulatory to inhibitory with increasing concentrations of caldesmon [Ishikawa et al., 1991: J. Biol. Chem. 266:21784–21790]. The N‐terminal (myosin‐binding) fragment and the C‐terminal (actin‐binding) fragment were purified from digests of caldesmon. The effects of the myosin‐binding fragment and the actin‐binding fragment on the interaction were stimulatory and inhibitory, respectively, indicating that stimulatory and inhibitory domains are localized in the myosin‐binding domain and actin‐binding domain of caldesmon, respectively. The effect of the myosin‐binding fragment on the interaction was exclusively stimulatory when the interaction was challenged by caldesmon, both at lower and higher concentrations. However, the actin‐binding fragment had no effect on the interaction at lower concentrations and inhibited the interaction at higher concentrations. Thus, the stimulatory effect of caldesmon that is observed at lower concentrations can be explained by the hypothesis that the stimulatory effect of the myosin‐binding domain predominates over the inhibitory effect of the actin‐binding domain when the concentration of caldesmon is low. With uncleaved caldesmon, we also emphasized the role of the myosin‐binding domain in the stimulation as follows; the stimulatory effect of caldesmon became obscured when binding of caldesmon to myosin was competed by the exogenous caldesmon‐binding fragment of myosin. © 1994 Wiley‐Liss, Inc.
Interactions among endoplasmic reticulum, microtubules, and retrograde movements of the cell surface
Authors: Terasaki Mark , Reese Thomas S.
Published: February 2005

Relationships among the endoplasmic reticulum (ER), microtubules, and bead movements on the cell surface were investigated in the thin peripheral region of A6 cells, a frog kidney cell line. ER tubules were often aligned with microtubules, as shown by double‐labeling with DiOC6(3) and anti‐tubulin in fixed cells. In living cells stained with DiOC6(3) and observed in time lapse, there were frequent extensions, but few retractions, of ER tubules. In addition, there was a steady retrograde (towards the cell center) movement of all of the ER at ∼0.3 μm/min. Since microtubules are often aligned with the ER, microtubules must also be moving retrogradely. By simultaneous imaging, it was found that the ER moves retrogradely at the same rate as aminated latex beads on the cell surface. This indicates that the mechanisms for ER and bead movement are closely related. Cytochalasin B stopped bead and ER movement in most of the cells, providing evidence that actin is involved in both retrograde movements. The ER retracted towards the cell center in nocodazole while both ER and microtubules retracted in taxol. Time lapse observations showed that for both drugs, the retraction of the ER is the result of retrograde movement in the absence of new ER extensions. Presumably, ER extensions do not occur in nocodazole because of the absence of microtubules, and do not occur in taxol because taxol‐stabilized microtubules move retrogradely and there is no polymerization of new microtubule tracks for ER elongation. © 1994 Wiley‐Liss, Inc.

This Article is a US Government work and, as such, is in the public domain in the United States of America.
Myosin I localizes to the midbody region during mammalian cytokinesis
Authors: Breckler Jennifer , Burnside Beth
Published: February 2005

During cytokinesis, daughter cells are cleaved in two by the constriction of an actin‐rich contractile ring which encircles the equator of the dividing cell. Filamentous myosin II is present in the contractile ring and necessary for constriction of the furrow, as shown in several cell types [Satterwhite and Pollard, 1992: Curr. Opin. Cell Biol. 4:43–52]. However, no functional role nor distinctive localization has been previously identified for non‐filamentous “unconventional” myosins, such as myosin I, during cytokinesis. Using antibodies to adrenal medullary myosin I, we report that myosin I is localized in 3T3 fibroblasts to the mid‐equatorial plane during late‐cytokinesis, as well as to the polar edges as previously described in ameboid cells [Fukui et al., 1989: Nature 341:328–331]. Confocal microscopy revealed that myosin I is concentrated at the midbody region in a nearly continuous transverse disk, extending from the cortical region of the furrow through the midbody itself. These findings suggest that, in addition to the accepted role of filamentous myosin II in constriction of the contractile ring, non‐filamentous myosin I might contribute to motile events occurring late in cytokinesis. © 1994 Wiley‐Liss, Inc.
PMA and calcium ionophore induce myosin and F‐actin rearrangement during histamine secretion from RBL‐2H3 cells
Authors: Ludowyke Russell I. , Kawasugi Kazuo , French Peter W.
Published: February 2005

Rat basophilic leukemia (RBL‐2H3) cells undergo morphological and cytoskeletal changes during antigen‐induced secretion of allergic mediators. The exact role these changes play in the process of secretion is unclear. Using confocal microscopy we now show that PMA + A23187 causes extensive F‐actin rearrangements during secretion of [3H] 5‐HT. We also describe for the first time the association of myosin with F‐actin during this secretory process. In unstimulated cells, myosin and F‐actin are concentrated at the plasma membrane with no evidence of stress fibres. Upon addition of PMA or A23187, both F‐actin and myosin are rearranged into membrane ruffles and discrete aggregations (foci), followed by the formation of parallel stress fibres located on the ventral membrane. This is in contrast to reports in other cell types in which PMA has been described as causing the disruption of F‐actin stress fibres. The time course of secretion coincides with the formation of the foci and ruffles whilst the stress fibres form after the majority of secretion has occurred. These changes are accompanied by a 40% decrease in cell height and a two‐fold increase in cell spreading and they occur in the absence of extracellular calcium but are inhibited by the protein kinase C inhibitor, Bisindolylmaleimide, which also inhibits secretion. The formation of myosin‐decorated stress fibres, foci, and ruffles is not sufficient to cause secretion, as PMA alone induces these changes without any secretion. The relevance of actin and myosin rearrangements for the regulation of secretion is discussed. © 1994 Wiley‐Liss, Inc.
Regulation of motility and cytoskeletal organization of rat bladder carcinoma cells by cyclic AMP
Authors: Morton Diane M. , Tchao Ruy
Published: February 2005

Cyclic AMP (cAMP) has been implicated in the regulation of movement of certain cultured cell types. We have studied the effects of cAMP on epithelial cell motility using serum‐free NBT‐II cells, derived from a rat bladder carcinoma. The random movement of these cells on type I collagen was reduced upon elevation of intracellular cAMP by several means and this effect was reversible. Alterations in the organization of the cytoskeletal proteins F‐actin and α‐actinin occurred concurrently with the reduction in motility, and the arrangement of these proteins resembled that seen in non‐motile cells on glass. In addition, pretreatment of cells with KT5720, a cAMP‐dependent protein kinase (PKA)‐specific inhibitor, prevented the dibutyryl cAMP‐induced reduction in cell movement as well as the associated cytoskeletal changes. These results suggest that elevation of PKA is responsible for the observed effects on cell motility and cytoskeletal reorganization and demonstrate a role for PKA in the regulation of cell motility in this system. © 1994 Wiley‐Liss, Inc.
Tubulin gene expression during growth and maturation of leaves with different developmental patterns
Authors: Hellmann Andreas , Meyer Claudius U. , Wernicke Wolfgang
Published: February 2005

Changes in the tubulin‐protein and ‐poly(A)+RNA contents were monitored by means of Western and Northern blot analyses, respectively, during growth and maturation of leaves of a dicotyledonous (tobacco) and monocotyledonous (barley) plant. It was recently argued from immunofluorescence and preliminary biochemical data that the density of microtubular networks and concomitantly the tubulin content are distinctly reduced after cessation of cell growth in leaves [Jung et al., 1993]. The results presented now confirm and extend this view. There appeared to be clear differences between the monocot and the dicot: (1) the loss of tubulin during leaf development was much slower in the dicot than in the monocot leaves (within months instead of days); (2) the degree of loss was more dramatic in the monocot leaf and only very low threshold levels of tubulin were retained in fully differentiated tissues; and (3) the loss of tubulin in the monocot leaf tissue appeared to be correlated with the decrease in the mRNA content, whereas the high level of tubulin‐RNA in fully differentiated or even almost senescent dicot leaves indicated a gene expression control at the posttranscriptional level.

The comparatively rapid and very distinct tubulin‐protein and ‐RNA disappearance during development of the monocot leaf tissues confirm at the molecular level that differentiation proceeds much faster and is much more determinative in these leaves, as was postulated from histological and physiological data. The differences in the behaviour of the microtubular cytoskeleton perhaps even reflect the differences in the ability of the differentiated leaf cells to dedifferentiate, i.e., to establish new sets of microtubules and to reenter the mitotic cell cycle, e.g., during would response, tumour induction or in vitro culture. © 1995 Wiley‐Liss, Inc.
Kinesin does not support the motility of zinc‐macrotubes
Authors: Ray Sanghamitra , Wolf Sharon G. , Howard Jonathon , Downing Kenneth H.
Published: February 2005

Moving along a microtubule, kinesin follows a course parallel to the protofilaments; but it is not known whether kinesin binds exclusively on a single protofilament. The presence of zinc during tubulin polymerization induces sheets where neighboring protofilaments are antiparallel. If kinesin could support the motility of these zinc‐sheets, then the binding site for a kinesin molecule would be limited to a single protofilament.

Kamimura and Mandelkow [1992: J. Cell Biol. 118:865–75] reported that kinesin moves along zinc‐sheets. We found that zinc‐sheets grown under their conditions often had a microtubule‐like structure along one edge. We confirmed the possibility that the motility observed by Kamimura and Mandelkow [1992: J. Cell Biol. 118:865–75] is attributed to the microtubule‐like structure rather than the zinc‐sheet.

To resolve the question of whether kinesin can recognize an antiparallel protofilament lattice, we investigated the kinesin‐mediated motility of zinc‐macrotubes. At higher free zinc concentrations, zinc‐sheets roll up as macrotubes, free of edges. In the presence of 10 m̈M taxol and 100 nM free Zn2+ at pH 6.8, the samples were shown by electron microscopy to contain only macrotubes. Under these buffer conditions, kinesin could bind strongly to axonemal doublets in the presence of AMP‐PNP, and generate motility in the presence of ATP, but kinesin did not bind to nor move the macrotubes. This shows that kinesin cannot bind efficiently to nor move on the anti‐parallel lattice; it is possible (though not necessary) that the groove between two parallel protofilaments is required for kinesin's motility. © 1995 Wiley‐Liss, Inc.
Monoclonal anti‐dipeptide antibodies cross‐react with detyrosinated and glutamylated forms of tubulins
Authors: Kuriyama Ryoko , Levin Andrew , Nelson David , Madl James , Frankfurther Anthony , Kimble Mary
Published: February 2005

Two monoclonal antibodies, GLU‐1 and A1.6, raised against γ‐L‐glutamyl‐L‐glutamic acid dipeptide (Glu‐Glu) and Ca2+ ‐dependent ATPase from Paramecium, respectively, recognized the dipeptide Glu‐Glu sequence. Whereas the antibodies immunofluorescently stained very few, if any, cytoskeletal fibers in cultured mammalian cells, almost all interphase as well as mitotic spindle microtubules became visible after treatment of cells with carboxypeptidase A. Immunoblot analysis demonstrated intense cross‐reaction of the antibodies to the α‐tubulin subunit. α‐Tubulin isotypes produced as fusion proteins in bacteria were labeled by both the antibodies only when the proteins did not contain a tyrosine residue at the C terminus, indicating that GLU‐1 and A1.6 specifically recognize the detyrosinated from of α‐tubulin. When microtubule protein purified from brain was probed, not only α‐but also, to a lesser extent, β‐tubulin were revealed by the dipeptide antibodies. A synthetic tripeptide YED containing one glutamyl group linked to the second residue of the peptide via the γ position was also recognized by the antibodies. Since this peptide sequence corresponds to the amino acid sequence of polyglutamyated class IIIβ isotype at amino acid position 437 to 439, it is suggested that GLU‐1 and A1.6 are able to recognize the glutamylated form of β‐tubulin. These results indicate that the C‐terminal Glu‐Glu sequence displays strong antigenicity, and the antibodies recognize the sequence present in the C terminus of the detyrosinated form of α‐tubulin and the glutamyl side chain of β‐tubulin. Particularly strong immunoreaction was detected with ciliary and flagellar microtubules; thus, stable axonemal microtubules appear to be rich in post‐translationally modified tubulin subunits. © 1995 Wiley‐Liss, Inc.
Chemoreceptor‐mediated polymerization and depolymerization of actin in hair bundles of sea anemones
Authors: Watson Glen M. , Roberts Julia
Published: February 2005

Hair bundles located on tentacles of sea anemones are morphodynamic mechanoreceptors employed to regulate discharge of nematocysts into swimming prey. Activation of chemoreceptors for N‐acetylated sugars is known to induce anemone hair bundles to elongate while shifting discharge to lower frequencies matching those produced by calmly swimming prey. In the continued presence of N‐acetylated sugars, activation of proline receptors is known to induce hair bundles to shorten while shifting nematocyst discharge to higher frequencies presumed to correspond to movements produced by wounded, struggling prey. In the present study, N‐acetylneuraminic acid (NANA) causes stereocilia to become more intensely fluorescent in confocal optical sections of phalloidin‐stained specimens, suggesting that receptors for N‐acetylated sugars initate processes to increase the density of F‐actin within stereocilia. Computer analysis of electron micrographs is consistent with this interpretation for large diameter stereocilia but not for small diameter stereocilia. In the continued presence of NANA, proline causes flurescence intensity of phalloidin to decrease to or below control levels. DNaseI uniformly stains large diameter stereocilia, suggesting that these stereocilia contain a pool of G‐actin. Fluorescence intensity of DNaseI in stereocilia is significantly less bright in specimens exposed to NANA alone than in specimens exposed to proline in the continued presence of NANA. It appears that whereas activated receptors for NANA induce G‐actin to polymerize in large diameter stereocilia, activated receptors for proline induce F‐actin to depolymerize, restoring G‐actin pools. © 1995 Wiley‐Liss, Inc.
Organization and structure of actin filament bundles in Listeria‐infected cells
Authors: Zhukarev Vladimir , Ashton Francis , Sanger Jean M. , Sanger Joseph W. , Shuman Henry
Published: February 2005

During its motion inside host cells, Listeria monocytogenes promotes the formation of a column of actin filaments that extends outward from the distal end of the moving bacterium. The column is constructed of short actin filaments that polymerize at the bacteria‐column interface. To get a measure of filament organization in the column, Listeria grown in cultured PtK2 cells were studied with steady state fluorescence polarization, confocal microscopy, and whole cell intermediate voltage electron microscopy. Although actin filament ordering was higher in nearby stress fibers than in the Listeria‐associated actin, four distinct areas of ordering could be observed in fluorescence polarization ratio images of bacteria: (1) the surface of the bacteria, (2) the cytoplasm next to the bacteria, (3) the outer shell of the actin column, and (4) the core of the column. Filaments were preferentially oriented parallel to the long axis of the column with highest ordering along the long axis of the bacterial surface and in the shell of the tail. The lowest ordering was in the core (where filaments are possibly also shorter with respect to the cup and the shell), whereas in the adjacent cytoplasm, filaments were oriented perpendicular to the column. A mutant of Listeria that can polymerize actin around itself but cannot move intracellularly does not have its actin organized along the bacterial surface. Thus the alignment of the actin filaments along the bacterial surfaces may be important for the intracellular movement. These conclusions are also supported by confocal microscopy and whole mount electron microscopic data that also reveal that actin filaments can be deposited asymmetrically around the long axis of the bacteria, a distribution that may affect the direction of motility of Listeria monocytogenes inside infected cells. © 1995 Wiley‐Liss, Inc.
Dynein inner arm heavy chain identification in cAMP‐activated flagella using class‐specific polyclonal antibodies
Authors: Stephens R. E. , Prior G.
Published: February 2005

While studying cAMP‐dependent dynein α‐heavy chain phosphorylation, we found previously [Stephens and Prior, 1992: J. Cell Sci. 103:999–1012] that high salt extraction of sperm flagella from the mussel Mytilus edulis or the clam Spisula solidissima removed most visible dynein arms, accompanied by an amount of Mg+2‐ATPase that correlated with the mass of dynein α‐and β‐heavy chains removed. However, although almost devoid of ATPase activity, such extracted axonemes retained one third of the heavy chain mass as two sets of electrophoretically‐distinct, vanadate‐cleavable, non‐phosphorylated proteins. To explore the nature of these dynein‐like proteins, antibodies to the α‐ and β‐heavy chains were blot affinity‐purified from a rabbit antiserum raised against gradient‐purified Spisula 18‐20S flagellar outer arm dynein. Although able to recognize common epitopes of the opposite chain type, neither the α‐nor the β‐heavy chain antibody recognized the tightly‐bound proteins in either species, proving that they are immunologically distinct. While the β‐antibody recognized its heavy chain homolog in gill cilia, the α‐antibody did not, demonstrating immunological distinction between flagellar and ciliary dynein α‐heavy chains. Immunization of a mouse with nitrocellulose strips containing one of the two tightly‐bound Spisula flagellar proteins produced an antiserum that cross‐reacted with each tightly‐bound protein in both species and also recognized α‐ and β‐heavy chains. The anti‐molluscan serum cross‐reacted strongly with sea urchin sperm flagellar dynein B‐, C‐, and D‐bands, considered to be inner arm components, but not with sea urchin outer arm α‐ or β‐heavy chains. These data indicate that the electro‐phoretically and immunologically distinct, tightly‐bound proteins of molluscan flagella are inner arm dynein heavy chains. © 1995 Wiley‐Liss, Inc.
β‐Tubulin mutation suppresses microtubule dynamics in vitro and slows mitosis in vivo
Authors: Sage Carleton R. , Davis Ashley S. , Dougherty Cynthia A. , Sullivan Kevin , Farrell Kevin W.
Published: February 2005

Microtubule (MT) dynamics vary both spatially and temporally within cells and are thought to be important for proper MT cellular function. Because MT dynamics appear to be closely tied to the guanosine triphosphatase (GTPase) activity of β‐tubulin subunits, we examined the importance of MT dynamics in the budding yeast S. cerevisiae by introducing a T107K point mutation into a region of the single β‐tubulin gene, TUB2, known to affect the assembly‐dependent GTPase activity of MTs in vitro. Analysis of MT dynamic behavior by video‐enhanced differential interference contrast microscopy, revealed that T107K subunits slowed both the growth rates and catastrophic disassembly rates of individual MTs in vitro. In haploid cells tub2‐T107K is lethal; but in tub2‐T107K/tub2‐590 heterozygotes the mutation is viable, dominant, and slows cell‐cycle progression through mitosis, without causing wholesale disruption of cellular MTs. The correlation between the slower growing and shortening rates of MTs in vitro, and the slower mitosis in vivo suggests that MT dynamics are important in budding yeast and may regulate the rate of nuclear movement and segregation. The slower mitosis in mutant celis did not result in premature cytokinesis and cell death, further suggesting that cell‐cycle control mechanisms “sense” the mitotic slowdown, possibly by monitoring MT dynamics directly. © 1995 Wiley‐Liss, Inc.
“Geometric clutch” hypothesis of axonemal function: Key issues and testable predictions
Authors: Lindemann Charles B. , Kanous Kathleen S.
Published: February 2005

No Abstratct.
Flagellar quiescence response in sea urchin sperm induced by electric stimulation
Authors: Shingyoji Chikako , Takahashi Keiichi
Published: February 2005

To investigate the mechanism of the flagellar quiescence in sperm, we examined the effect of electric stimulation of individual spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. Stimulation with a suction electrode attached to the sperm head elicited a flagellar quiescence response, in which the sperm showed a typical cane‐shaped bend in the proximal region of the flagellum when the electrode was used as anode. Cathodic stimulation also induced quiescence, but was much less effective than anodic stimulation. During the quiescence response, which lasted for 1–3 s, no new bend was initiated, and subsequently the flagellum resumed normal beating. The quiescence response required the presence of Ca2+ (>2 mM) in sea water, and was inhibited by Co2+ and La3+. At low Ca2+ concentrations (2–5 mM), the angle of the cane‐shaped bend was smaller than that at 10 mM Ca2+; thus the angle of the cane‐shaped bend, characteristic of the quiescence response is dependent on Ca2+ concentration. These results suggest membrane, followed by an influx of Ca2+ into the flagellum through Ca2+ channels. The increase in Ca2+ concentration within the flagellum affects the amount of sliding and thus produces a cane‐shaped proximal bend of various angles, white inhibiting both the propagation of the proximal bend (principal bend) and the formation of a new reverse bend.
Experimental manipulation of γ‐tubulin distribution in arabidopsis using anti‐microtubule drugs
Authors: Liu Bo , Palevitz Barry A. , Joshi Harish C.
Published: February 2005

γ‐Tubulin‐specific antibodies stain the microtubule (Mt) arrays of Arabidopsis suspension cells in a punctate or patchy manner. During division, staining of kinetochore fibers and the phragmoplast is extensive, except in the vicinity of the plus ends at the metaphase plate and cell plate. γ‐Tubulin localization responds to low levels of colchicine, with staining receding farther toward the minus (pole) ends of kinetochore fibers. At higher drug concentrations, γ‐tubulin also associates with abnormal Mt foci as well as with the surface of the daughter nuclei facing the phragmoplast. During UV‐induced recovery from colchicine, γ‐tubulin increases along the presumptive minus ends of mitotic Mts as well as the phragmoplast near the daughter nuclei. With CIPC, immunostaining is concentrated around the centers of focal Mt arrays in multipolar spindles. In the presence of taxol, Mts are more prominent but the mitotic apparatus and phragmoplast are abnormal. As with CIPC, γ‐tubulin is concentrated at focal arrays. Increased punctate staining is also present in interphase arrays, with fluorescent dots often located at the ends of Mts. These results support a preferential association between γ‐tubulin and Mt minus ends, but are also consistent with more general binding along the walls of Mts. Thus, minus ends (and Mt nucleation sites) may be present throughout plant Mt arrays, but γ‐tubulin may also serve another function, such as in structural stabilization.
Exogenous gelsolin binds to sarcomeric thin filaments without severing
Authors: Gonsior Sabine , Hinssen Horst
Published: February 2005

We have investigated the binding of gelsolin to thin myofilaments in situ and their stability against severing. Differentiated myotubes from chicken skeletal muscle containing cross‐striated myofibrils were permeabilized with Triton X‐100 and incubated with gelsolin. Immunoflurorescence microcopy localized both endogenous and exogenous gelsolin in the I‐Z‐I‐regions of the sarcomers. The staining pattern suggested a binding of the exogenous gelsolin along the entire length of the thin filaments. This binding was Ca2+ dependent, but gelsolin was not removed after subsequent addition of EGTA. The fluorescence staining for actin remained unchanged after gelsolin incubation, indicating that thin filaments in cross‐striated myofibrils were resistant to the severing action of gelsolin, in contrast to the microfilaments in stress fibers. After extraction of the permeabilized cells with high ionic strength to remove tropomyosin and myosin, gelsolin stell bound along the entire thin filament and the actin pattern also remained unchanged. After Triton X‐100 permeabilization and high ionic strength extraction, the giant protein nebulin was found to be still present as a myofibrillar component. Gelsolin treatment after high salt extraction affected neither actin nor nebulin in the thin filaments. We therefore conclude that nebulin confers the gelsolin resistance to the sarcomeric actin filaments.
Overexpression of an epitope‐ tagged β‐tubulin in Chinese hamster ovary cells causes an increase in endogenous α‐tubulin synthesis
Authors: Gonzalez‐Garay Manuel L. , Cabral Fernando
Published: February 2005

A Chinese hamster β‐tubulin cDNA, engineered to express a 9 amino acid epitope from the influenza hemagglutinin antigen (HA), was transfected into Chinese hamster ovary (CHO) cells. The recombinant protein (HAβ1‐tubulin) appeared to behave normally by the following criteria: immunofluorescence indicated that HAβ1‐tubulin incorporated into all classes of interphase and spindle microtubules as well as microtubule organizing centers. The sensitivity of the cells expressing HAβ1‐tubulin to Colcemid and taxol was unchanged. A 210 kD microtubule associated protein (MAP) remained associated with microtubules that incorporate HAβ1‐tubulin. The synthesis of both endogenous β‐tubulin and HAβ1‐tubulin was repressed by colchicine. The HAβ1‐tubulin incorporated into microtubules to the same extent as the endogenous β‐tubulin, and the overall extent of microtubule assembly in transfected cells was unchanged. Finally, trasfected cells had normal growth rates and morphologies. When effects on endogenous tubulin production were measured, it was found that expression of the HAβ1‐tubulin reduced the synthesis of endogenous wild‐type β‐tubulin but increased the synthesis of α‐tubulin. At steady state, a small increase in total tubulin consistent with the increased synthesis of α‐tubulin was found. The results indicate that expression of excess exogenous β‐tubulin perturbs the synthesis of endogenous α‐tubulin in a manner that is not easily explained by current models of tubulin regulation. The changes in tubulin synthesis along with degradation of excess tubulin subunits may reflect mechanisms that exist to ensure coordinate levels of α‐ and β‐tubulin for assembly. © 1995 Wiley‐Liss, Inc.
Molecular environment of ZO‐1 in epithelial and non‐epithelial cells
Authors: Howarth Andrew G. , Stevenson Bruce R.
Published: February 2005

We previously reported the expression of ZO‐1 in cell types that do not form tight junctions. Here we compare the molecular environments of ZO‐1 in epithelial cells, primary cultures of astrocytes and in the non‐epithelial S180 sarcoma cell line. ZO‐1 co‐localizes with a subset of actin filament in all cell types. In astrocytes, ZO‐1 is found concentrated in discrete bands at points of cell‐cell contact. Indirect immunofluorescent microscopy shows that these bands of ZO‐1 co‐localize with the adherens junction proteins vinculin and α‐actinin, and with the antigen recognized by a pan‐cadherin antibody. In contrast, ZO‐1 in S180 cells, which exhibit limited cell‐cell interactions, is diffusely distributed over the plasma membrane, with concentrations in lamellipodia where actin filaments accumulate. ZO‐1 does not co‐localize with vinculin at focal adhesions in this cell type. Analysis of ZO‐1 immunoprecipitation profiles from different cell types, performed under conditions previously demonstrated to maintain interactions between ZO‐1, ZO‐2 and p130 from the MDCK epithelial cell line, show that the proteins which co‐precipitate with ZO‐1 vary with cell type. Precipitation of polypeptides at 165 kDa, potentially ZO‐2, and 65 kDa occurs in both a mouse kidney tubule epithelial cell line and the non‐epithelial S180 cells. No proteins specifically associate with ZO‐1 immunoprecipitated from astrocytes. Spectrin, α‐actinin, vinculin and cadherin are not detected in immunoblots of ZO‐1 immunoprecipitates from any cell type. © 1995 Wiley‐Liss, Inc.
Regulatory role of nucleotides in axonemal function
Authors: Kinoshita Satoko , Miki‐Noumura Taiko , Omoto Charlotte K.
Published: February 2005

Axonemal sliding involves both sliding velocity and the extent of sliding, that is how many doublets slide. It is clear that axonemes cannot beat if all doublets were to slide simultaneously, thus sliding extent is important. Using the turbidimetric assay of sliding disintegration of Tetrahymena axonemes, we examined the sliding extent and the effect of ADP, ATP, and ATP analogs on the sliding extent. Of course, ATP is necessary to produce sliding disintegration, but ATP alone did not produce extensive sliding disintegration. The addition of ADP allowed greater extent of sliding disintegration. The additions of higher ATP concentration even in the presence of ADP inhibited sliding disintegration. We also observed sliding disintegration using ribose‐modified ATP analogs, anthraniloylATP, and methylanthraniloylATP. The extent of sliding disintegration was proportional to the analog concentration. Thus in contrast to ATP, higher analog concentration was not inhibitory. These results indicate that high ATP concentration acts to inhibit the extent of sliding disintegration and that ADP relieves this inhibition. We propose a model in which the affinity of multiple cooperative active sites are regulated by binding of ATP or ADP to a regulatory site. This model provides a mechanism by which nucleotides regulate the extent of sliding necessary for effective axonemal bending. © 1995 Wiley‐Liss, Inc.
Deletion of amino acids from the carboxy‐terminal end of actin
Authors: Xia Dong , Peng Isaac
Published: February 2005

A series of deletions was made from the C‐terminal end of actin by inserting termination codons into a full length cDNA of human α‐skeletal muscle actin. These included deletions of 2, 3, 10, 20, 30, and 40 amino acids. The cDNA clones were transcribed and the resulting mRNA were translated in vitro using 35S‐labeled methionine. The 35S‐labeled actin and actin mutants were then tested for the ability to coassemble with carrier actin, bind DNAse I, bind myosin S‐1, bind a 27 kDa proteolytic fragment of α‐actinin, and incorporate into myofibrils in vitro. Removal of the C‐terminal two or three amino acids did not grossly alter the properties of actin tested. Deletion of an additional 7 amino acids (10 amino acids total) significantly decreased coassembly, binding to DNAse I, and incorporation into myofibrils, but did not dramatically reduce binding to myosin S‐1 or the 27 kDa fragment of α‐actinin. Deletion of 20 or more amino acids virtually abolished all normal actin function tested. By examining the structure of actin, we propose that the effect of removing residues 356–365 is due to the important role Trp356 plays in maintaining hydrophobic bonds between three non‐contiguous segments of actin. We also suggest that removal of residues 366–372 adversely affected the structure or orientation of the DNAse I binding loop and that this change can account for defects in actin binding to DNAse I, coassembly with wild type actin, and incorporation into myofibrils. © 1995 Wiley‐Liss. Inc.
Over‐expression of smooth muscle caldesmon in mouse fibroblasts
Authors: Surgucheva Irina , Bryan Joseph
Published: February 2005

Caldesmon is an actin, calmodulin, tropomyosin, and myosin binding protein implicated in the regulation of actomyosin interactions. We have invesigated the effect of overexpression of the higher molecular weight smooth muscle isoform of caldesmon on mouse L cell physiology. Mouse L(TK−) cell were transfected stably with plasmids carrying the TK+ gene and a full length human smooth muscle caldesmon cDNA under control of the adenovirus major late promoter. Two clones displaying four and eight times the level of the endogenous mouse high molecular weight caldesmon were isolated. These cells acquire a distinct phenotype characterized by an altered morphology, including an increased number of processes and larger area due to enhanced cell spreading, and a significantly slower growth rate than that of untransfected control cells, or cells transfected with the TK+ gene alone. The majority of the overexpressed caldesmon appears to be active and localized on cytoskeleton structures as determined by detergent lysis. Immuno‐fluorescence analysis of the clones revealed that the caldesmon is localized as punctate staining on stress‐fibers and in membrane ruffles. The immunofluores‐cence images suggest that caldesmon overexpressing cells have more total filaments than control cells. The effects of excess caldesmon on cell mobility are ambiguous: one clone displayed increased motility compared to the control, while the motility of the second clone was decreased relative to the control. © 1995 Wiley‐Liss, Inc.
Analysis of the interdependent localization of vimentin and microtubules in neoplastic myoepithelial cells
Authors: Jaeger Ruy G. , Jaeger Márcia M. M. , Araújo Vera C. , Kachar Bechara
Published: February 2005

Salivary gland neoplastic myoepithelial cells in culture form very thin cytoplasmic processes in which the vimentin network is well dispersed. These vimentin filaments can be individually visualized by immunofluorescence. In this study, we have analyzed the role of microtubules in the distension and organization of the vimentin filament network found in these cells. We find that vimentin filaments colocalize along microtubules; however, a significant number of filaments can also be found in microtubule‐free domains. Additionally, vimentin filaments are absent from large domains of microtubule inhibitor nocodazole did not cause any retraction of the distended vimentin network. This observation suggests that the structural integrity of microtubules is not important for the stability of the vimentin network. Combining procedures for transient disruption of vimentin filaments and microtubules we observed that, in the absence of microtubules, the vimentin network could reassemble in the perinuclear region but was unable to extend toward the cell periphery. The dispersion of vimentin filaments to the peripheral regions of the cytoplasm could only be observed upon microtubule reassembly. This indicates that microtubules are not required for the stability of the vimentin network, but the dispersion of vimentin filaments to the peripheral cytoplasm depends on active interactions with microtubules. © 1995 Wiley‐Liss, Inc.
Microtubule‐associated movement of mitochondria and small particles in Acanthamoeba castellanii
Authors: Baumann Otto , Murphy Douglas B.
Published: February 2005

Using video‐enhanced differential interference microscopy and digital image processing, we have observed organelle motility in Acanthamoeba castellanii. In amoebae taken from cultures in rapid growth phase, mitochondria and small particles moved over distances of several microns and at an average velocity of ∼2 μ/s. Mitochondrial motility was verified by intensified fluorescence microscopy of cells that were labeled in vivo with the DNA‐binding dye DAPI or the mitochondria‐specific dye Mito Tracker. We further studied the role of microtubules (MTs) in the translocation of cell organelles. Double‐labelling of fixed cells bules with mitochondrial markers (anti‐F1β antibody, Mito Tracker) and cytoskeletal markers (anti‐tubulin antibody, rhodamine‐phalloidin) demonstrate that the mitochondria colocalize with MTs in the subcortical cell area and are excluded from the F‐actin‐rich cell cortex. Colchicine treatment resluted in an almost complete depolymerization of MTs and an inhibition of organelle motility. Moreover, we have directly visualized MTs in vivo in flattened amoebae. Mitochondria and small particles moved along the MTs in a bidirectional mode at an average velocity of ∼1 μm/s. We conclude that the observed movement of mitochondria and small particles in Acanthamoeba castellanii mainly occurs via microtubules and associated motor proteins. © 1995 Wiley‐Liss, Inc.
Vinculin localization and actin stress fibers differ in thyroid cells organized as monolayers or follicles
Authors: Yap Alpha S. , Stevenson Bruce R. , Waters Michael J. , Keast Janet R. , Manley Simon W.
Published: February 2005

In epithelial cells interactions between the actin cytoskeleton and cell‐cell junctions regulate paracellular permeability and partcipate in morphogenesis. We have studied the relationship between supracellular morphology and actin‐junction interactions using primary cultures of porcine thyroid cells grown either as three‐dimensional follicles or as open monolayers. Regardless of morphology, thyroid cells assembled occluding and adhesive junctions containing ZO‐1 and E‐cadherin, respectively, and showed F‐actin staining in apical microvilli and a perijunctional ring. In monolayers, actin stress fibers were also observed in the apical and basal poles of cells, where they terminated in the vinculin‐rich zonula adherens and in cell‐substrate focal adhesions, respectively. Surprisingly, we were unable to detect vinculin localization in follicular cells, which also did not form stress fibers. Immunoblotting confirmed significantly greater vinculin in triton‐insoluble fractions from monolayer cells compared with follicular cells. Incubation of monolayers with 8 chloro(phenylthio)‐cyclic AMP decreased the level of immunodetectable vinculin in the zonula adherens, indicating that junctional incorporation of vinculin was regulated by cyclic AMP. In monolayer cultures, cytochalasin D (1 μM) caused actin filaments to aggregate associated with retraction of cells from one another and the disruption of cell junctions. Despite morphologically similar perturbations of actin organization in follicular cultures treated with cytochalasin D, junctional staining of ZO‐1 and E‐cadherin was preserved and cells remained adherent to one another. We conclude that in cultured thyroid cells structural and functional associations between actin filaments and cellular junctions differ depending upon the supracellular morphology in which cells are grown. One important underlying mechanism appears to be regulation of vinculin incorporation into adhesive junctions by cyclic AMP. © 1995 Wiley‐Liss, Inc.
Studies on spontaneous formation of 1D nanocrystals of silicon carbide
Authors: Huczko A. , Lange H. , Bystrzejewski M. , Rűmmeli M. H. , Gemming T. , Cudziło S.
Published: February 2005

Novel nanostructures have been formed spontaneously during the thermolysis of mixtures containing halogenohydrocarbons and Si‐related compounds as a reducing agent in a calorimetric bomb. Elemental analysis, XRD (X‐Ray Diffraction), EELS (Electron Energy Loss Spectroscopy), EDX (Energy Disspersive X‐ray spectroscopy), IR (InfraRed spectroscopy), Raman spectroscopy, HR SEM (High Resolution Scanning Electron Spectroscopy) and TEM (Transmission Electron Spectroscopy) were used to determine the chemical composition and structural features of the products. They contain well‐crystallized elongated 1D single crystals of SiC nanofibres, coated with a thin SiO2 amorphous layer, and crystallized carbon nanoparticles. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)