bluuebunny/crossref_metadata_2025 huggingface dataset:
Contributions of the β‐subunit to spectrin structure and function
Authors: Coleman Thomas R. , Fishkind Douglas J. , Mooseker Mark S. , Morrow Jon S.
Published: February 2005

The three avian spectrins that have been characterized consist of a common α‐subunit (240 kD) paired with an isoform‐specific β‐subunit from either erythrocyte (220 or 230 kD), brain (235 kD), or intestinal brush border (260 kD). Analysis of avian spectrins, with their naturally occurring “subunit replacement” has proved useful in assessing the relative contribution of each subunit to spectrin function. In this study we have completed a survey of avian spectrin binding properties and present morphometric analysis of the relative flexibility and linearity of various avian and human spectrin isoforms. Evidence is presented that, like its mammalian counterpart, avian brain spectrin binds human erythroid ankyrin with low affinity. Cosedimentation analysis demonstrates that (1) avian erythroid protein 4.1 stimulates spectrin‐actin binding of both mammalian and avian erythrocyte and brain spectrins, but not the TW 260/240 isoform, (2) calpactin I does not potentiate actin binding of either TW 260/240 or brain spectrin, and (3) erythrocyte adducin does not stimulate the interaction of TW 260/240 with actin.

In addition, a morphometric analysis of rotary‐shadow images of spectrin isoforms, individual subunits, and reconstituted complexes from isolated subunits was performed. This analysis revealed that the overall flexibility and linearity of a given spectrin heterodimer and tetramer is largely determined by the intrinsic rigidity and linearity of its β‐spectrin subunit. No additional rigidity appears to be imparted by noncovalent associations between the subunits. The scaled flexural rigidity of the most rigid spectrin analyzed (human brain) is similar to that reported for F‐actin.
cAMP‐independent and cAMP‐dependent protein phosphorylations by isolated goldfish xanthophore cytoskeletons: Evidence for the association of cytoskeleton with a carotenoid droplet protein
Authors: Palazzo Robert E. , Lynch Thomas J. , Taylor John D. , Tchen T. T.
Published: February 2005

Triton‐insoluble cytoskeleton of nonpigment cells has bound protein kinase that phosphorylates, with or without added cAMP, tubulins and the intermediate filament proteins p60, p56, p53, and p45a to give multiple charge variants. In the absence of 8‐Br‐cAMP, Triton‐insoluble cytoskeletons from xanthophores also phosphorylate p60, p56, and p45a, but not p53; tubulin phosphorylation may also be reduced. In the presence of 8‐Br‐cAMP, p53, as well as several other peptides, are phosphorylated. One of these latter peptides was identified as the carotenoid droplet (pigment organelle) protein p57, whose phosphorylation and dephosphorylation precede pigment dispersion and aggregation respectively (Lynch et al.: J. Biol. Chem. 261:4204–4211, 1986). The amount of pp57 produced depends on the state of pigment distribution in the xanthophores used to prepare the cytoskeletons for labeling. With cytoskeletons from xanthophores with aggregated pigment, pp57 is a major labeled phosphoprotein seen in two‐dimensional gels. With cytoskeletons prepared from xanthophores with dispersed pigment, the yield of labeled pp57 is greatly reduced (by at least 90%). Together with earlier results, we propose that, in the aggregated state, p57 serves to bind carotenoid droplets to the cytoskeletons, most likely the microtubules. The significance of other cAMP‐dependent phosphorylation reactions is unknown but may be related to cAMP‐induced cytoskeleton rearrangement in intact xanthophores.
Evidence that intermediate‐like filaments are found in the micronucleus of Paramecium bursaria during prophase
Authors: Lewis Larry M.
Published: February 2005

The micronuclear spindle apparatus in Paramecium bursaria was studied by electron microscopy during prophase, metaphase, and anaphase of the first meiotic division. During prophase, the spindle apparatus consists mostly of intermediate‐like filaments, relatively few spindle microtubules, and unique cone‐shaped structures termed microlamellae. Microlamellae join the ends of chromosomes to the fibrous elements of the spindle. The capacity to preserve the intermediate‐like filaments is largely dependent upon the use of collidine buffer during fixation. In contrast, during metaphase and anaphase, microtubules are the dominant fibrous element of the spindle. The microtubules interact with chromosomes during these phases by joining to true kinetochores. Neither treatment with cytochalasin B or fixation with a low concentration of osmium tetroxide affects the development of intermediate filaments during prophase. Because intermediate‐like filaments are abundant during prophase and microtubules are more common during metaphase and anaphase, the structural differences may reflect differences in the mechanisms for chromosome movement.
Long‐term observation of cultured cells by interference‐reflection microscopy: Near‐infrared illumination and Y‐contrast image processing
Authors: Zand Martin S. , Albrecht‐Buehler Guenter
Published: February 2005

Interference‐reflection microscopy (IRM) is the only method presently available with which to visualize cell‐substratum adhesions in living tissue culture cells continuously for long periods of time without the use of fluorescent markers (Curtis: J. Cell Biol. 20:199–215, 1964; Izzard and Lochner: J. Cell Sci. 21:129–159, 1976). This method utilizes approximately 1% of the incident illumination to produce the IRM image (Verschueren: J. Cell Sci. 75:279–301, 1985) and so far has required the use of high‐intensity light sources in the visible spectral range (400–800 nm). Unfortunately, visible light of this intensity and spectral range induces marked changes in the behavior and morphology of motile fibroblasts, including cessation of locomotion. In contrast, the present paper reports that continuous observations of live cells in IRM for periods of up to 8 hours are possible if the illuminating light is in the red to near‐infrared range (650–950 nm) and without any observable change in normal cell morphology or behavior. In addition, we describe how the technique of Y‐contrast image processing can be applied to IRM images to create a three‐dimensional image of the ventral cell surface topography.
Mitoskelin: A mitochondrial protein found in cytoskeletal preparations
Authors: Price Maureen G. , Gomer Richard H.
Published: February 2005

A 70 kD protein, which we have named mitoskelin, is highly enriched in cytoskeletal preparations from bovine cardiac muscle. Mitoskelin has three main variants with isoelectric points between 5.6 and 5.8. Immunoblotting with polyclonal antibodies directed against mitoskelin shows that, like intermediate filament proteins, the majority of mitoskelin resists solubilization from a myocardial homogenate by a series of extraction solutions ranging from very low salt to 0.6 M KI buffers and by 0.1–1% Nonidet P‐40 detergent. By double‐label immunofluorescence on cells and tissues, mitoskelin is colocalized with the mitochondrial marker cytochrome c oxidase. Mitoskelin is associated with the inner membranes of mitochondria as shown by immunoelectron microscopy and immunoblotting Immunological cross‐reactivity and similarities of molecular weight, pI, distribution, and chromatographic properties indicate that mitoskelin is the 70 kD component of complex I (NADH: ubiquinone oxidoreductase), a portion of the mitochondrial oxidative phosphorylation system. No function or activity has yet been demonstrated for the 70 kD component of the 25‐polypeptide complex I. Dialysis against physiological buffers allows purified, urea‐solubilized mitoskelin to form 10 nm wide filamentous structures that do not closely resemble intermediate filaments. These results suggest the exciting possibility that mitochondria may contain a membrane‐associated filamentous skeleton.
Rapid growth cone translocation on laminin is supported by lamellipodial not filopodial structures
Authors: Kleitman N. , Johnson M. I.
Published: February 2005

To determine the relationship between growth cone structure and motility, we compared the neurite extension rate, the form of individual growth cones, and the organization of f‐actin in embryonic (E21) and postnatal (P30) sympathetic neurons in culture. Neurites extended faster on laminin than on collagen, but the P30 neurites were less than half as long as E21 neurites on both substrata. Growth cone shape was classified into one of five categories, ranging from fully lamellipodial to blunt endings. The leading margins of lamellipodia advanced smoothly across the substratum ahead of any filopodial activity and contained meshworks of actin filaments with no linear f‐actin bundles, indicating that filopodia need not undirlie lamellipodia. Rapid translocation (averaging 0.9–1.4 μm/min) was correlated with the presence of lamellipodia; translocation associated with filopodia averaged only 0.3–0.5 μm/min. This relationship extended to growth cones on a branched neurite where the translocation of each growth cone was dependent on its shape. Growth cones with both filopodial and lamellipodial components moved at intermediate rates. The prevalence of lamellipodial growth cones depended on age of the neurites; early in culture, 70% of E21 growth cones were primarily lamellipodial compared to 38% of P30 growth cones. A high percentage of E21 lamellipodial growth cones were associated with rapid neurite elongation (1.2 mm/day), whereas a week later, only 16% were lamellipodial, and neurites extended at 0.5 mm/day. Age‐related differences in neurite extension thus reflected the proportion of lamellipodial growth cones present rather than disparties in basic structure or in the rates at which growth cones of a given type moved at different ages. Filopodia and lamellipodia are each sufficient to advance the neurite margin; however, rapid extension of superior cervical ganglion neurites was supported by lamellipodia independent of filopodial activity.
Selective reduction of anaphase B in quinacrine‐treated PtK1 cells
Authors: Armstrong L. , Snyder Judith A.
Published: February 2005

Quinacrine, an acridine derivative which competitively binds to ATP binding sites, has previously been shown to cause the reorganization of metaphase spindle microtubules (MTs) due to changes in interactions of non‐kinetochore microtubules (nkMTs) of opposite polarity (Armstrong and Snyder: Cell Motil. Cytoskeleton 7:10–19, 1987). In the study presented here, mitotic PtK1 cells were treated in early anaphase with concentrations of quinacrine ranging from 2 to 12 μM to determine energy requirements for chromosome motion. The rate and extent of chromosome‐to‐pole movements (anaphase A) were not affected by these quinacrine treatments. The extent of anaphase B (kinetochore‐kinetochore separation) was reduced with increasing concentrations of quinacrine. Five micromolar quinacrine reduced the extent of kinetochore‐kinetochore separation by 20%, and addition of 12 μM quinacrine reduced the kinetochore‐kinetochore separation by 40%. To determine the role of nkMTs in anaphase spindle elongationquinacrine‐treated metaphase cells were treated with hyperosmotic sucrose concentrations, and spindle elongation was measured (Snyder et al.: Eur J. Cell Biol. 39:373–379, 1985). Metaphase cells treated with 2–10 μM concentrations of quinacrine for 2–5 min reduced spindle lengths by 10–50% prior to 0.5 M sucrose treatment for 5 min. This treatment showed a significant reduction in the ability of sucrose to induce spindle elongation in cells pretreated with quinacrine. As spindle length and birefringence was reduced by quinacrine treatment, sucrose‐induced elongation was concomitantly diminished. These data suggest that quinacrine‐sensitive linkages are necessary for anaphase B motions. Reduction in these linkages and/or MT length in the nkMT continuum may reduce the ability of the nkMTs to hold compression at metaphase. This form of energy is thought to drive a significant proportion of normal anaphase B in PtK1 cells and sucrose‐induced metaphase spindle elongation.
Nonuniform behavior of multiple isoactins in the same cell is a cell‐dependent phenomenon
Authors: Shires Ann K. , Rubenstein Peter A.
Published: February 2005

The functional significance of multiple isoactins in the same cell is still not understood. To address this question, we examined the response of smooth muscle and cardiac muscle α‐isoactins to a serial extraction procedure applied to both muscle and nonmuscle cell types. We compared these extraction results with results obtained with the β‐ and γ‐nonmuscle actin isoforms from the same cells. In differentiated BC3H1 nonfusing muscle cells (smooth muscle α‐isoactin), in human rhabdomyosarcoma cells (cardiac α‐isoactin), and in chick skeletal muscle cells (cardiac α‐isoactin), different fractions were found selectively enriched in either the nonmuscle or the muscle‐specific actin isoforms compared with their relative abundance in whole cell extracts. Conversely, when these same isoactins were examined either in undifferentiated BC3H1 cells or in mouse nonmuscle cells stably transfected with a cardiac α‐isoactin gene, no enrichment of these isoforms above their relative abundance in whole cell extracts was observed. These results indicate that within the muscle or muscle‐like cells examined, the different actin isoforms were either selectively utilized or localized. These results further show that isoactin‐specific responses observed were apparently related to the cell type in which they were found and not to differences in inherent physical properties such as solubility of the different isoactins examined.
Structure and function of the cytoskeleton in heliozoa: I. Mechanism of rapid axopodial contraction in Echinosphaerium
Authors: Ando Motonori , Shigenaka Yoshinobu
Published: February 2005

In the heliozoan Echinosphaerium, rapid axopodial contraction often occurs during food capturing. In morphological studies, it has been considered that rapid contraction is caused by conformation is change of the X‐body. We determined that reaction sites or receptors for anion exchange resin, which can induce rapid contraction, were located in every region of the axopodium. However, the probability of locating sites where contraction was induced was lower in the middle region than in the distal region of the axopodium. In cases in which contraction was not induced by resin placed in the middle region of the axopodium, so‐called bead formation was induced instead. At the fine structural level, only granulated forms of the X‐body were observed in this beading region. These results suggest that rapid contraction results from disassembly of axonemal microtubules and the simultaneous contraction of the X‐body and that bead formation represents a stage when only the X‐body contracts. Furthermore, effects of metabolic inhibitors and Ca2+ channel blockers revealed that contraction of the X‐body did not depend on the supply of adenosine triphosphate, but on Ca2+ influx. Ultrastructural observations revealed that the mass of the granulated X‐body was aggregated into the proximal region of the axopodium, suggesting that the X‐body might be associated with the undercoat of axopodial membrane or the axonemal microtubules themselves.
Binding of microtubule‐associated proteins (MAPs) to rat brain mitochondria: A comparative study of the binding of MAP2, Its microtubule‐binding and projection domains, and tau proteins
Authors: Jancsik Veronika , Filliol Dominique , Felter Simone , Rendon Alvaro
Published: February 2005

Two major brain microtubule‐associated proteins (MAPs), MAP2 and tau, were found to be able to bind to purified rat brain mitochondria. The apparent dissociation constants of the binding of thermostable 32P‐labeled MAP2 and tau are 0.9 ± 0.04 × 10−7 and 3.8 ± 0.7 × 10−7 M, respectively. 32P‐labeled MAP2 and tau bound to the mitochondria can be displaced by phosphorylated, nonradioactive MAP2. The binding parameters of MAP2 prepared without heat treatment and those of the thermostable MAP2 were of the same order of magnitude. Microtubule‐binding and projection domains of MAP2 were obtained by chymotryptic digestion of rat brain microtubules (Vallee, Proc. Natl. Acad. Sci. USA, 77:3206‐3210, 1980). Displacement studies with these two domains show that MAP2 bound to mitochondria can be displaced by the microtubule‐binding domain, whereas the projection domain does not displace MAP2. The two domains of MAP2 bind to the mitochondria with similar affinity constants; however, the Bmax for the projection domain was 10 times and 35 times lower than the Bmax of the binding of the intact MAP2 and the microtubule‐binding domain, respectively. Chymotryptic digestion of MAP2 bound to the mitochondria yielded peptide fragments with molecular masses similar to those obtained by the digestion of MAP2 bound to the microtubules. The fragments corresponding to the projection domain were released into the extramitochondrial supernatant, whereas the fragments originating from the microtubule‐binding domain remained bound to the mitochondria. These results suggest that MAP2 binds to mitochondria preferentially via its microtubule‐binding domain.
External mechanical control of the timing of bend initiation in sea urchin sperm flagella
Authors: Eshel Dan , Gibbons I. R.
Published: February 2005

The movement parameters of a sea urchin sperm flagellum can be manipulated mechanically by applying various modes of periodic vibrations to the sperm head held by suction in the tip of a micropipette. The beat frequency of the flagellum readily synchronizes with the frequency of the externally imposed lateral vibration, and the plane of flagellar bending waves adapts itself to the plane of the pipette vibration (Gibbons et al., J. Cell Biol. 101:270a, 1985; Nature 325: 351–352, 1987). In this study, we observed the particular effects of external asymmetric forces on flagellar beating parameters by vibrating the micropipette holding the sperm head in a transverse sawtooth‐like motion composed of a rapid effective stroke and a slower recovery stroke, while keeping the vibration frequency constant. The results demonstrate that the timing of bend initiation within the flagellar beat cycle can be controlled mechanically by changing the time point within the vibration cycle at which the micropipette changes its direction of motion. A switch in the sidedness of the asymmetric movement of the micropipette produces dramatic changes in the profiles of bend growth in the basal 5 μm of the flagellum but has almost no effect on the asymmetry or other parameters of bending in the mid‐ and distal regions of the flagellum. Our results suggest that elastic strain within the basal region of the flagellar structure may play a more significant role in the process of bend initiation than has been realized heretofore.
Rise of internal Ca2+ accompanies the initiation of trout sperm motility
Authors: Cosson Marie Paule , Billard Roland , Letellier Lucienne
Published: February 2005

The initiation of motility of trout spermatozoa is inhibited by the presence of millimolar concentrations of external K+, but external Ca2+ might also be implicated in this control as it has been shown to antagonize the K+ inhibition of motility [S.M. Baynes et al.: J. Fish. Biol., 19:259–267, 1981]. The present work aimed to investigate internal Ca2+ levels during the motility phase of trout spermatozoa. Internal Ca2+ concentrations were monitored by the fluorescent quinoline Ca2+‐indicator, “Quin‐2” [R. Y. Tsien: Nature 290:527–529, 1981]. Trout spermatozoa were loaded with Quin‐/ under conditions that gave efficient intracellular hydrolysis of Quin‐2 and that did not impair the ability of loaded spermatozoa to initiate movement. The beat frequencies, cell velocities, and flagellar asymmetries of sperm movement were not significantly modified by the presence of the internal dye. Upon initiation of flagellar movement, an increase of the internal Quin‐2 fluorescence was observed that reflected a sixfold increase of the free Ca2+ concentration. The free Ca2+ remained elevated after the cessation of movement. The variation of fluorescence was completed within 40 seconds, whereas the initiation of motility was nearly instantaneous, and the total duration of flageliar beating lasted for about 80–100 seconds (measurements at 11°C). The increase in the internal free Ca2+ concentration is completed after the initiation of flagellar beating but its occurrence correlates with that of sperm movement. Fluorescence increase was not observed in the presence of 40 mM K+, a condition in which spermatozoa did not initiate flagellar beating. In the presence of the Ca2+ channel blocker desmethoxyverapamil, neither sperm motility nor fluorescence increases were observed, which suggested that the increase of internal free Ca2+ was produced by a flux of external Ca2+ into the cell rather than by a mobilization of internal Ca2+ stores.
Molecular analysis of the alpha and beta dynein genes of Chlamydomonas reinhardtii
Authors: Mitchell David R.
Published: February 2005

We previously cloned portions of the alpha and beta dynein heavy chain genes of Chlamydomonas reinhardtii by screening a genomic expression library with monoclonal antibodies (Williams et al.: Journal of Cell Biology 103:1–11, 1986). Here we provide further evidence of the identity of these clones and describe the selection of adjacent regions from a large insert genomic library. Southern blots indicate that only a single copy of each gene is present in the Chlamydomonas genome, while Northern blots show that both heavy chains are encoded by 13.5 kilobase mRNAs and that the corresponding transcription units each span approximately 20 kilobase‐pairs of genomic DNA. No similarities were detected between restriction maps of the alpha and beta dynein genes, but extensive regions of sequence similarity were identified by the cross‐hybridization of cloned gene fragments.
Immunofluorescence evidence for cytoskeletal rearrangement accompanying pigment redistribution in goldfish xanthophores
Authors: Walker Gary R. , Taylor John D. , Tchen T. T.
Published: February 2005

Immunofluorescence and phase‐contrast microscopic studies of goldfish xanthophores with aggregated or dispersed pigment show two unusual features. First, immunofluorescence studies with anti‐actin show punctate structures instead of filaments. These punctate structures are unique for the xanthophores and are absent from both goldfish dermal no‐pigment cells and a dedifferentiated cell line (GEM‐81) derived from a goldfish xanthophore tumor. Comparison of immunofluorescence and phase‐contrast microscopic images with electron microscopic images of thin sections and of Triton‐insoluble cytoskeletons show that these punctate structures represent pterinosomes with radiating F‐actin. The high local concentration of actin around the pterinosomes results in strong localized fluorescence such that, when the images have proper brightness for these structures, individual actin filaments elsewhere in the cell are too weak in their fluorescence to be visible in the micrographs. Second, whereas immunofluorescence images with anti‐tubulin show typical patterns in xanthophores with either aggregated or dispersed pigment, namely, filaments radiating out from the microtubule organizing center, immunofluorescence images with anti‐actin or with anti‐intermediate filament proteins show different patterns in xanthophores with aggregated versus dispersed pigment. In cells with dispersed pigment, the punctate structrues seen with anti‐actin are relatively evenly distributed in the cytoplasm, and intermediate filaments appear usually as a dense perinuclear band and long filaments elsewhere in the cytoplasm. In cells with aggregated pigment, both intermediate filaments and pterinosomes with associated actin are largely excluded from the space occupied by the pigment aggregate, and the band of intermediate filaments surrounds not only the nucleus but also the pigment aggregate. The patterns of distribution of the different cytoskeleton components, together with previous results from this laboratory, indicate that formation of the pigment aggregate depends at least in part on the interaction between pigment organelles and microtubules. The possibility that intermediate filaments may play a role in the formation/stabilization of the pigment aggregate is discussed.
Clathrin‐coated membrane: A distinct membrane domain in acetylcholine receptor clusters of rat myotubes
Authors: Pumplin David W. , Bloch Robert J.
Published: February 2005

We have used antibodies to clathrin light chains in immunocytochemical studies of acetylcholine receptor (AChR) clusters of cultured rat myotubes. Immunofluorescence and ultrastructural experiments show that clathrin is present in coated pits and in large plaques of coated membrane. Coated membrane plaques are spatially and structurally distinct from AChR‐rich membrane domains and the bundles of microfilaments that are also present in AChR clusters. Clusters contain a relatively constant amount of clathrin light chain protein, which is not dependent on the amount of AChR. Clathrin plaques remain after AChR domains are disrupted by azide, or after microfilament bundles are destabilized by cytochalasin D. Extraction of myotubes with saponin removes clathrin without disrupting AChR domains. Thus, clathrin plaques, microfilament bundles, and AChR‐rich domains are independently stabilized.
Actin‐dependent carotenoid droplet dispersion in permeabilized cultured goldfish xanthophores
Authors: Yu Fu‐Xin , Taylor John D. , Tchen T. T.
Published: February 2005

Organelle translocations are essential cellular processes. Although much progress has been made with regards to microtubule‐dependent organelle translocations, little is known about actin‐dependent organelle translocation(s) except cytoplasmic streaming in Nitella. On the other hand, there is indirect evidence that actindependent organelle translocation may be involved in secretion. We now present evidence that the dispersion of the pigment organelles carotenoid droplets in goldfish xanthophores is apparently actin dependent and that this process may be related to secretory processes. We show that, in digitonin‐permeabilized goldfish xanthophores, the pigment organelles can be induced to disperse by a combination of cAMP, ATP, and xanthophore cytosol. This induced dispersion is inhibited by DNase I, phalloidin, or anti‐actin, but not by anti‐tubulin or anti‐intermediate filament proteins, suggesting a dependence on F‐actin. Since the dispersion of carotenoid droplets and secretion both involve outward translocation of organelles, we tested the possibility that cytosols of secretory tissues have similar activity. Such activity was indeed found in different tissues, apparently in parallel with the secretory activity of the tissues, suggesting that pigment dispersion in xanthophores and some secretory processes may share a common component.
Diffuse kinetochores and holokinetic anaphase chromatin movement during mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC‐20
Authors: Rieder Conly L. , Bowser Samuel S. , Cole Richard , Rupp Gerald , Peterson Andrew , Alexander Stephen P.
Published: February 2005

Mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC‐20 was examined by light microscopy of living and fixed cells. During early prometaphase the numerous small (0.30–3.0‐μm) chromosomes appear as discrete units that lack a primary constriction. However, by late prometaphase the chromosomes are tightly packed at the spindle equator and are no longer clearly resolvable as individuals. When viewed from the side the metaphase chromatin appears as a 2–3‐μm wide band that spans the width of the spindle; when viewed from the pole it appears as a fenestrated disk. The metaphase chromatin splits at anaphase into two sister chromatin plates, each of which exhibits holokinetic poleward movement, i.e., all parts of each plate move as a single unit with the same velocity. In many early‐to‐mid anaphase cells the separating sister plates are connected by chromatin‐containing bridges that break as anaphase progresses. Ultrastructural analyses of serial thick and thin sections from cells fixed by conventional, OsO4/KFeCN, or high pressure rapid freezing methods, reveal that by metaphase all of the chromosomes are interconnected to form a large, irregularly shaped fenestrated disk of chromatin. Similar analyses reveal that adjacent chromatids remain interconnected throughout anaphase. Each disk of metaphase and anaphase chromatin contains numerous kinetochores recessed within its polefacing surface. Kinetochores consist of a fine, faintly staining fibrillar material arranged along the chromatin surface as thin (0.1–0.3 μm dia.) rods varying considerably (0.15–2.3 μm) in length. From these observations we conclude that the polycentric metaphase chromatin of A. constricta, and its holokinetic behavior during anaphase, arises from the aggregation or cohesion of smaller prometaphase chromosomes, each of which contains a single, diffuse kinetochore.
Motion of individual human spermatozoa, both normal and lacking the outer dynein arms, during a continuous temperature rise
Authors: Auger J. , Serres Catherine , Feneux Danielle
Published: February 2005

The effect of increasing temperature from 22–25°C to 37°C on various motion characteristics of individual normal human spermatozoa and spermatozoa lacking the outer dynein arms (LODA) was studied by using a new automatic microscopic tracking method. It was found that: (1) The curvilinear velocity (Vc, measured between 1–3 sec) of both normal and LODA spermatozoia, fluctuated more or less intensely between spermatozoa; this fluctuation was not thermodependent. (2) The average Vc in the two groups of spermatozoa increased with the rise in temperature at a similar rate (1μm/sec/°C), but LODA spermatozoa had an initial Vc lower than that of normal spermatozoa (12.5 ± 5.3 μm/sec and 34.2 ± 8.2 μm/sec, respectively). (3) The profile of the Vc increase associated with the temperature rise was different for the two groups of spermatozoa: for LODA spermatozoa it was linear between 25–37°C, whereas for normal spermatozoa a plateau was reached at about 31°C. (4) Various patterns of trajectory were found for both normal and LODA spermatozoa; these patterns were unrelated to temperature. However, LODA spermatozoa had more linear trajectories than normal spermatozoa. (5) Plots derived from reaction rate theory showed that the activation enthalpy, δH‡ was a function of the increase of Vc for both normal and LODA spermatozoa, but that δH‡ was higher for LODA spermatozoa.
Cytoskeletal dynamics in rabbit synovial fibroblasts: II. Reformation of stress fibers in cells rounded by treatment with collagenase‐lnducing agents
Authors: Aggeler Judith
Published: February 2005

Modulation of the synthesis and secretion of extracellular matrix proteins and matrix‐degrading metalloproteases by rabbit synovial fibroblasts in an important model system for studying the control of tissue‐specific gene expression. Induction of collagenase expression is correlated with changes in cell shape and actin filament distribution, but the role of the cellular cytoskeleton in the sustained synthesis and secretion of metalloproteases has not been closely examined. When cells were allowed to respread after rounding by trypsin or cytochalasin, two known metalloprotease inducers, reformation of stress fibers was observed within 2 h in the presence of serum. In the absence of serum, trypsin‐treated cells did not respread substantially, even after 24 h in culture. In contrast, cytochalasin‐treated cells recovered almost as rapidly in the absence as in the presence of serum, showing reformation of well‐formed microfilament bundles within 30 min of drug removal, especially at the spreading cell edges. High resolution electron‐microscopic views of detergent‐extracted cytoskeletons confirmed the rapid rebundling of peripheral microfilaments. Acrylamide‐treated cells fell between these two extremes, spreading slowly in the absence of serum, but almost as rapidly as cytochalasin‐treated cells in its presence. Reestablishment of normal intermediate filament distribution generally lagged slightly behind actin for all treatments, and intermediate filaments always appeared to spread back into the cellular cytoplasm within the confines of the reforming peripheral microfilament bundles. No obvious interaction between these two cytoskeletal elements was observed after any treatment, and no specific role for intermediate filaments in modulating gene expression in these cells is suggested by these results. The serum dependence displayed after trypsin or acrylamide treatment may be due to the disturbances in fibronectin synthesis observed in these cells and is consistent with evidence that both induction and sustained expression of matrix‐degrading metalloprotease may involve signals transduced through plasma membrane matrix receptors (integrins).
Differential effects of gelsolins on tissue culture cells
Authors: Huckriede Anke , Füchtbauer Annette , Hinssen Horst , Chaponnier Christine , Weeds Alan , Jockusch Brigitte M.
Published: February 2005

Gelsolins, prepared from a number of different sources, showed similar severing activity on F‐actin in vitro or on stress fibers of detergent‐extracted cells but differed in their effects on actin in stress fibers of microinjected cells. When human gelsolin isolated from plasma was injected into cells in a Ca++‐containing buffer, stress fibers were degraded, the cellular morphology was changed, and numerous actin patches appeared. These effects were particularly striking when the Ca++‐insensitive N‐terminal proteolytic fragment of this gelsolin was injected. By contrast, Ca++‐sensitive gelsolins isolated from human platelets, pig stomach smooth muscle and pig plasma showed no comparable activity. Furthermore, the Ca++‐independent N‐terminal proteolytic fragments prepared from these gelsolins also had no effect despite their in vitro actin severing activity. Most striking was the finding that human plasma gelsolin expressed in E. coli did not degrade stress fibers, in contrast to the same protein isolated from plasma; nor was there any stress fiber disruption observed with the N‐terminal half of human gelsolin expressed in Escherichia coli.

The different behavior of these gelsolins in cells cannot be explained by sequence diversity between plasma and cytoplasmic forms, nor by variability in the Ca++ sensitivity of the preparations. It suggests the presence of factors, as yet unidentified, that may regulate gelsolin activity in the cytoplasm of living cells and discriminate between gelsolins of different origin. Such discrimination could be achieved as a result of post‐translational modification of the gelsolin; only in this way can differences between apparently identical proteins isolated from human plasma and expressed in E. coli be reconciled.
The expression and posttranslational modification of a neuron‐specific β‐tubulin isotype during chick embryogenesis
Authors: Lee Michael K. , Tuttle Jeremy B. , Rebhun Lionel I. , Cleveland Don W. , Frankfurter Anthony
Published: February 2005

Five β‐tubulin isotypes are expressed differentially during chicken brain development. One of these isotypes is encoded by the gene cβ4 and has been assigned to an isotypic family designated as Class III (βIII). In the nervous system of higher vertebrates, βIII is synthesized exclusively by neurons. A βIII‐specific monoclonal antibody was used to determine when during chick embryogenesis cβ4 is expressed, the cellular localization of βIII, and the number of charge variants (isoforms) into which βIII can be resolved by isoelectric focusing. On Western blots, βIII is first detectable at stages 12–13. Thereafter, the relative abundance of βIII in brain increases steadily, apparently in conjunction with the rate of neural differentiation. The isotype was not detectable in non‐neural tissue extracts from older embryos (days 10–14) and hatchlings. Western blots of protein separated by two‐dimensional gel electrophoresis (2D–PAGE) reveal that the number of βIII isoforms increases from one to three during neural development. This evidence indicates that βIII is a substrate for developmentally regulated, multiple‐site posttranslational modification. Immunocytochemical studies reveal that while cβ4 expression is restricted predominantly to the nervous system, it is transiently expressed in some embryonic structures. More importantly, in the nervous system, immunoreactive cells were located primarily in the non‐proliferative marginal zone of the neural epithelia. Regions containing primarily mitotic neuroblasts were virtually unstained. This localization pattern indicates that cβ4 expression occurs either during or immediately following terminal mitosis, and suggests that βIII may have a unique role during early neuronal differentiation and neurite outgrowth.
Ca/Ba/Sr‐induced conformational changes of ciliary axonemes
Authors: Tamm R. Sidney , Tamm Signhild
Published: February 2005

Macrocilia of the ctenophore Beroë undergo Ca/Ba/Sr‐dependent activation of ciliary beating and microtubule sliding disintegration [Tamm, J. Comp. Physiol. A163:23–31. 1988a: Tamm, Cell Motil. Cytoskeleton 11:126–138, 1988b; Tamm, Cell Motil. Cytoskeleton 12:104–112, 1989: Tamm and Tamm, Proc. Natl. Acad. Sci. U.S.A. 86:6987–6991, 1989]. Here we report that detergent‐extracted macrocilia show an ATP‐independent conformational change in response to high concentrations of Ca. Ba. or Sr ions. When applied locally by iontophoresis, these ions induce a rapid planar curvature of the distal end of the macrociliary shaft, followed by a slower relaxation to the rest position. Tip curling occurs in a direction opposite to the physiological Ca/Ba/Sr response. When applied uniformly in the bath, a threshold concentration of 10−1 M Sr is required to induce curling of the tip, and the distal ends remain curved. Calmodulin antagonists do not inhibit the tip curling response.

Previous workers found that Ca induces changes in the helical shape of isolated doublet microtubules [Miki‐Noumura and Kamiya, Exp. Cell Res. 97:451–453, 1976: Miki‐Noumura and Kamiya. J. Cell Biol. 81:355–360, 1979; Takasaki and Miki‐Noumura. J. Mol. Biol. 158:317–324, 1982] and sperm axonemes [Okuno and Brokaw, Cell Motil. 1:349–362. 1981] and suggested that conformational changes in microtubules may play a role in Ca regulation of ciliary motility. We propose that the Ca/Ba/Sr‐induced curling of the macrociliary tip is due to similar helical changes of doublet microtubules, some of which in macrocilia are prevented from sliding by permanent connections (compartmenting lamellae) between adjacent axonemes within the shaft. Although the tip curling response does not appear to be directly relevant to the physiological Ca response of macrocilia, it provides a novel system for investigating Ca‐induced conformational changes of microtubules independent of dynein‐powered active sliding.
Effect of filamin and controlled linear shear on the microheterogeneity of F‐Actin/Gelsolin gels
Authors: Cortese Jorge Daniel , Frieden Carl
Published: February 2005

We have previously established [Cortese and Frieden, J. Cell Biol. 107:1477–1487, 1988] that actin gels formed under shear are microheterogeneous. In this study, the effect of cross‐linking (by chicken gizzard filamin), severing (by plasma gelsolin), and shear on actin microheterogeneity are investigated using fluorescence photobleaching recovery and video microscopy. We find that filamin and shear form microheterogeneous F‐actin:gelsolin gels by different mechanisms. Bundling of actin:gelsolin filaments by filamin can be explained by an increase in the apparent length of the filaments due to interfilament binding, resulting in a decrease of the polymer number concentration at which filaments organize into anisotropic phases. Some intrafilament binding of filamin to actin filaments may also be present, and those filaments coated with filamin immobilize more slowly than actin under the same polymerization conditions. The length of F‐actin/gelsolin filaments seems to be a major factor in controlling the extent of bundling relative to network formation. In contrast, the effect of shear on the microheterogeneity of actin:gelsolin filaments is consistent with our previous proposal that shear aligns actin filaments, allowing filament‐filament interactions and phase formation to occur. Short filaments are unable to organize into branched actin networks, but they can create large aggregates under low shear. Longer actin filaments will exist as networks with variable levels of branching and are less sensitive to shear. The effect of the intensity of a shear field on the spatial distribution of actin may involve a progressively more random orientation of actin molecules and bundles. A regular pattern develops across the sample at low shear rates (0.04–1.39 s−1), and becomes very irregular at higher shear rates (> 10 s−1). We suggest here that actin‐binding proteins and shear can control the transition between isotropic networks and anisotropic phases by their effect on apparent length and local filament concentration, and also that this transition can have substantial effects on the resistance of cells to mechanical stress.
Plants contain highly divergent actin isovariants
Authors: Gail McLean Barbara , Huang Shurong , McKinney Elizabeth Cohen , Meagher Richard B.
Published: February 2005

Actin protein isovariants have been identified in animals with distinct cytoplasmic or muscle specific patterns of expression. Analysis of vascular plant actin gene sequences suggests that an even greater diversity should exist within the plant actin protein families, but previous studies on plant proteins have not demonstrated the presence of multiple actin isovariants. Antibodies recognizing a conserved amino‐terminal plant actin peptide, a family of plant actin peptides from a variable region, and two monoclonal antibodies to conserved epitopes within animal actins were used to identify isovariants of soybean actin resolved by two‐dimensional isoelectric focusing (IEF) sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Approximately six to eight actin isovariants with pi values ranging from 5.1 to 5.8 have been identified from soybean hypocotyls. Stems, leaves, and roots with varying amounts of most isovariants present in all four organs. Acidic isovariants were present in much higher levels in leaves and stems. Antisera with λ‐class actin specificity detected a subset of three isovariants in all organs examined. One monoclonal and one antipeptide antisera are shown to react well with a wide variety of plant actin isovariants. Similar patterns of actin isovariants were detected in the distant angiosperms, Arabidopsis petunia, and maize. It is likely that many of these diverse classes of isovariants have been preserved throughout vascular plant evolution and reflect the ancient diversity within plant actin gene families. The extreme difference among isovariants implies the presence of a complex actin‐based cytoskeletal system in plants.
Transmembrane assemblage of the photoreceptor connecting cilium and motile cilium transition zone contain a common immunologic epitope
Authors: Horst Cynthia J. , Johnson Lincoln V. , Besharse Joseph C.
Published: February 2005

The photoreceptor connecting cilium bears a unique transmembrane assemblage which stably links cell surface glycoconjugates with the underlying axonemal cytoskeleton. Structural similarities between the photoreceptor connecting cilium and the transition zone of motile cilia suggests that this assemblage may also be present in motile cilia. Using a subcellular fraction enriched in detergent‐extracted photoreceptor axonemes, three high molecular mass glycoconjugates (425, 600, and 700 kD) were previously identified as potential components of the assemblage. Through oligosaccharide characterization and binding of a specific monoclonal antibody, we have verified the localization of the 425 kD glycoconjugate to the transmembrane assemblage. Binding of the lectin peanut agglutinin (PNA) to the 425 kD glycoconjugate on nitrocellulose blots, and to isolated detergentextracted axonemes, was assessed following treatment with the enzymes neuraminidase and O‐glycanase. Changes in binding to the 425 kD glycoconjugate precisely paralleled changes in binding to intact axonemes, supporting the hypothesis that the 425 kD glycoconjugate is a component of the transmembrane assemblage. Furthermore, the results suggest that the 425 kD glycoconjugate contains sialated galactose‐N‐acetylgalactosamine oligosaccharides which are Olinked to the protein backbone. To directly assess the distribution of the 425 kD glycoconjugate, we produced a monoclonal antibody directed against this glycoconjugate. The antibody, K26, recognizes only the 425 kD on transblots of the axoneme fraction. K26 immunoreactivity of intact axonemes is identical to that seen by PNA staining. K26 staining of isolated photoreceptors and whole retina is uniquely localized to the region of the connecting cilium. Thus, in the photoreceptor, the 425 kD is not only a component of the transmembrane assemblage but is also completely restricted to the connecting cilium.

Based on morphological similarities, the photoreceptor connecting cilium is thought to be homologous to the transition zone of the motile cilium. As such, we have stained oviduct epithelium with the K26 monoclonal antibody. Immunoreactivity is restricted to the region of the transition zone at the base of motile cilia. This demonstrates that the photoreceptor connecting cilium and motile cilium transition zone are immunologically related.
Dynamic changes of microtubule and actin structures in CV‐1 cells during electrofusion
Authors: Zheng Qiang , Chang Donald C.
Published: February 2005

To study the involvement of the cytoskeletal system in the fusion of animal cells, we examined the dynamic changes of cytoskeletal proteins during the various stages of cell fusion. CV‐l cells were fused by applying a radio‐frequency electrical pulse. Structural changes of microtubules (MTs) and F‐actin were monitored simultaneously by double‐label fluorescence microscopy. It was observed that in a few minutes after the initiation of cell fusion, MT bundles began to extend into the cytoplasmic bridges which were formed by fusing the membranes of neighboring cells. Later, a network of parallel MT bundles appeared between the adjacent nuclei of the fusing cells; such MT bundles may provide the mechanical links that are responsible for nuclear aggregation. The structural changes of Factin during cell fusion were more complicated. We observed many different patterns of actin distribution in the fusing cells, including some giant, ring‐shaped structures. Reorganization of actin is unlikely to be involved in the nuclear aggregation process. Instead, actin bundles condensed at the cell edges may help to widen the cytoplasmic bridges to allow merging of cellular contents between the fusing cells.
Reorganization of cytoskeletal and junctional proteins during cochlear hair cell degeneration
Authors: Raphael Yehoash , Altschuler Richard A.
Published: February 2005

Experiments were carried out to elucidate changes in cytoskeletal elements and intercellular junctions in the organ of Corti, when hair cells degenerate and phalangeal scars form. Hair cell damage was induced by exposing guinea pigs to high intensity noise. The spatial and temporal changes in the organization of micro‐filaments, intermediate filaments, and tight junction‐specific proteins were investigated using scanning and transmission electron microscopy and histochemistry. The results show that microfilaments, cytokeratins, adherens junctions, and tight junctions rearrange their distribution in damaged areas. From the temporal sequence of these changes it appears that phalangeal scars develop simultaneous with hair cell degeneration, and that the integrity of the luminal membranes in the organ of Corti is not interrupted. Each scar is formed by two supporting cells which expand and invade the sub‐apical region of the dying hair cell. This region becomes cytokeratin‐positive. The two supporting cells meet at the mid‐line of the scar, where a new junctional complex is formed. The junctional complex consists of tight junction and adherens‐type junction, but desmosomes are absent.