bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure and mass of mammalian respiratory ciliary outer arm 19S dynein
Authors: Hastie Annette T. , Marchese‐Ragona Silvio P. , Johnson Kenneth A. , Wall Joseph S.
Published: February 2005

Mammalian respiratory ciliary outer arm dyneins isolated as the major ATPase peak migrating at 19S on sucrose density gradients were examined by transmission electron microscopy of negatively stained samples and scanning transmission electron microscopy of unstained samples. The predominant discrete particle structure observed was composed of two globular heads apparently connected by amorphous or indistinct material. The heads were either circular or slightly elliptical of mean 13 ± 1×10 ± 2 nm dimensions. The mass of this structure averaged 1.22 ± 0.34 million daltons with the individual globular heads averaging 310 ± 77 kilodaltons (kD). Negative staining revealed that one or both of the globular heads often contained a central accumulation of stain measuring 2.5 ± 1 nm across. A second type of structure, appearing with lesser frequency in the 19S fraction than in the unfractionated dynein preparation loaded onto the sucrose gradient, was a single globular head of 13 ± 1×10 ± 2 nm often with 2 ± 1 nm centrally accumulated stain and with or without an appendage. This one‐headed particle thus resembled one‐half of the two‐headed particle. Mass measurements were lower, however, for isolated, single globular heads, averaging 220 ± 111 kD. A third type of particle observed was a ring‐like structure with 4 ± 1 nm centrally accumulated stain and without appendages. The ring structure was slightly larger in diameter, 14 ± 1 nm, and had a greater peripheral accumulation of negative stain than either of the one‐ or two‐headed particles, suggesting that it was not derived therefrom. Structures appearing as three globular heads were the least frequently observed. Mammalian respiratory ciliary outer arm 19S dynein is therefore a two‐headed structure and conforms to descriptions of outer arm dyneins from mammalian spermatozoa flagella and from invertebrate cilia or flagella.
The ciliary cycle during hyperpolarization‐induced activity: An analysis of axonemal functional parameters
Authors: Sugino Kazuyuki , Machemer Hans
Published: February 2005

Motor responses of the frontal cirri of the ciliate Stylonychia were recorded at the axial view of the ciliary base with high‐speed cinematography. Voltage‐clamp applying sustained hyperpolarizing voltage steps was used to explore the properties of the ciliary cycle modulated by the membrane potential. Upon hyperpolarization between – 1 and – 13 mV, a previously inactive frontal cirrus reoriented from a neutral posture and started beating so that the axis of the beating cone of a proximal cirral segment assumed an orientation near 100° (proceeding counterclockwise from posterior = 0°) and inclination near 60° (0° = perpendicular to the cell surface). The major beating amplitude was limited to about 150°. Increasing hyperpolarization increased the spatial polarity of the cycle (ratio of major over minor amplitude, from 2 to 2.4). Rates of the power stroke increased with hyperpolarizations up to – 4 mV but were consistently smaller than those of the return stroke during the ciliary cycle (ratio: 0.4 to 0.6; = temporal polarity). Comparison of different hypothetical beat forms (0‐shape, D‐shape, and egg‐shape) showed that the orientation‐time data are the major determinants of the angular velocity and rate of reorientation of the cilium during the cycle. Geometric transformation of these data led to descriptions of the cycle of a proximal ciliary segment in terms of active sliding velocities and rates of unidirectional sliding translocation between identified doublets. Three voltage‐sensitive functional parameters of the cilium—the inclination (which is noncyclic) and the rates of active sliding and sliding translocation (both of which are cyclic in nature)—are discussed as generating the spatial and temporal properties of the ciliary beat.
Effect of metabolic inhibitors on sucrose‐induced metaphase spindle elongation and spindle recovery
Authors: Snyder Judith Armstrong
Published: February 2005

Hyperosmotic sucrose treatment of metaphase PtK‐1 cells has been shown to produce a reversible concentration‐dependent effect on spindle elongation linked to a functional alteration in the connection of the chromosome to the spindle (Pover et al.: European Journal of Cell Biology 39:366–372, 1985). Spindle elongation, similar to that which occurs at anaphase B, is thought to be driven by the compression stored in the form of microtubule curvature in the nonkinetochore (nkMT) population of microtubules at metaphase (Snyder et al.: European Journal of Cell Biology 35:62–69, 1984 and 39:373–379, 1985). Addition of metabolic inhibitors to Ham's F‐12 salts with deoxyglucose (D/F‐12 medium) containing 0.4 M sucrose and 1 mM DNP does not within statistical error affect the rate and extent of sucrose‐induced spindle elongation; rates and extents are 60–75% of normal anaphase B motions. Electron microscopic analysis of metaphase cells treated with D/F‐12 medium and 0.4 M sucrose with 1 mM DNP demonstrates that spindle microtubules lose curvature and become straight in appearance, typical of microtubule organization in untreated anaphase cells. Sucrose‐treated cells released into D/F‐12 medium show a rapid reduction in spindle length; however, cells treated with either 0.4 M sucrose or 0.4 M sucrose and 1 mM DNP‐containing D/F‐12 medium and released into DNP‐containing D/F‐12 medium do not exhibit a significant reduction in spindle length. Electron microscopic analysis links changes in spindle length with microtubule/kinetochore associations. These data suggest that energy required for the initial phases of spindle elongation during anaphase is preloaded into the mitotic spindle by metaphase and does not require additional energy to be expressed as examined by sucrose‐induced spindle elongation in the presence of metabolic inhibitors. Second, energy is required to make or maintain (or both) functional chromosome associations with the spindle as measured by reduction in spindle length following sucrose removal.
Unique isoactins in the brush border of rat intestinal epithelial cells
Authors: Sawtell N. M. , Hartman A. L. , Lessard J. L.
Published: February 2005

The mammalian genome contains 20–30 genes encoding a family of actins. To date, however, only six proteins (four muscle and two nonmuscle isoforms) encoded by this multigene complex have been identified. We have isolated two actins from the brush border of rat intestinal epithelial cells that have isoelectric points and N‐terminal peptides characteristic of the cytoplasmic β‐ and γ‐actins. However, using a panel of actin‐specific monoclonal antibodies, we show that these actins contain a set of epitopes that distinguishes them from any of the known cytoplasmic or muscle isoforms. These unique actins share features of both the nonmuscle and muscle isoforms, suggesting that they represent an intermediate in the evolution of the specialized muscle actins.
Calcium‐dependent protein kinase is localized with F‐actin in plant cells
Authors: Putnam‐Evans Cindy , Harmon Alice C. , Palevitz Barry A. , Fechheimer Marcus , Cormier Milton J.
Published: February 2005

We recently purified a calcium‐dependent but calmodulin‐ and phospholipid‐independent protein kinase (CDPK) from cultured plant cells (Harmon et al.: Plant Physiology 83:830–837, 1987). A monoclonal antibody (mAb 3B9) directed against CDPK was used to localize this protein in Allium root cells and Tradescantia pollen tubes using immunofluorescence techniques. The mAb 3B9 staining pattern showed that CDPK is localized within a fibrous network in the cytoplasm resembling the normal interphase network of F‐actin. Treatment of tissue with 10 μM cytochalasin D (CD) prior to fixation abolished the staining pattern. Double‐localization experiments in which pollen tubes were first stained with mAb 3B9 and then with rhodamine‐phalloidin (RP) demonstrated that CDPK and F‐actin were colocalized. Monoclonal antibody 3B9 did not react with purified actin from rabbit muscle or Dictyostelium and did not bind to proteins corresponding to the Mr of actin in crude extracts of Allium root tips and Tradescantia pollen tubes.

CDPK did not phosphorylate purified rabbit muscle or Dictyostelium actin in vitro. Binding studies showed that CDPK (1) does not cosediment with actin filaments and (2) does not form a complex with G‐actin. The data indicate that although CDPK does not interact directly with actin, it may be associated with an actin‐binding protein and therefore could play a role in the regulation of the plant cytoskeleton.
A model for slow axonal transport and its application to neurofilamentous neuropathies
Authors: Blum J. J. , Reed M. C.
Published: February 2005

A model for slow axonal transport is developed in which the essential features are reversible binding of cytoskeletal elements and of soluble cytosolic proteins to each other and to motile elements such as actin microfilaments. Computer simulation of the equations of the model demonstrate that the model can account for many of the features of the SCa and SCb waves observed in pulse experiments. The model also provides a unified explanation for the increase and decrease of neurofilament transport rates observed in various toxicant‐induced neuropathies.
225‐Kilodalton phosphoprotein associated with mitotic centrosomes in sea urchin eggs
Authors: Kuriyama Ryoko
Published: February 2005

Protein phosphorylation during development of sea urchin eggs from fertilization to first cleavage was examined by labeling cells with specific antiphosphoprotein antibodies. Indirect immunofluorescence staining with monoclonal antithiophos‐phoprotein antibody (Gerhart et al.: Cytobios 43:335–347, 1985) has revealed that nuclei as well as centrosomes, kinetochores, and midbodies were specifically thiophosphorylated in developing eggs incubated with adenosine 5′‐O (3‐thiotriphosphate) (ATP‐γ‐S). The phosphorylation reaction required Mg2+ but was not dependent on cAMP or calmodulin in detergent‐extracted models. Centrosomes were purified by fractionation of isolated mitotic spindles with 0.5 M KCl extraction. The thiophosphoproteins were retained in the purified centrosomes and the antibody recognized a major 225‐Kd polypeptide on immunoblots. In an independent preparation, a monoclonal antiphosphoprotein antibody (CHO3) was found also to react with mitotic poles and stained a 225‐Kd polypeptide, confirming the centrosome specificity of this protein. Immunoelectron microscopy showed that the 225‐Kd thiophosphoprotein was found at mitotic poles associated with granules to which mitotic microtubules were directly attached. Unlike centrosomes in permeabilized eggs, those in isolated spindles could not be thiophosphorylated, possibly due to inactivation or loss of either phosphorylation enzymes or cofactors, or both, during isolation. The immunofluorescence labeling of thiophosphate could be inhibited by ATP and AMP‐PNP in a concentration‐dependent manner. Exogenous ATP could abolish thiophosphate‐staining more effectively when added with phosphatase inhibitors, suggesting a dynamic state in which centrosomal proteins are being phosphorylated and dephosphorylated in rapid succession by the action of protein kinase(s) and phosphatase(s).
Visualization of intermediate filaments in living cells using fluorescently labeled desmin
Authors: Mittal B. , Sanger J. M. , Sanger J. W.
Published: February 2005

Fluorescently labeled desmin was incorporated into intermediate filaments when microinjected into living tissue culture cells. The desmin, purified from chicken gizzard smooth muscle and labeled with the fluorescent dye iodoacetamido rhodamine, was capable of forming a network of 10‐nm filaments in solution. The labeled protein associated specifically with the native vimentin filaments in permeabilized, unfixed interphase and mitotic PtK2 cells. The labeled desmin was microinjected into living, cultured embryonic skeletal myotubes, where it became incorporated in straight fibers aligned along the long axis of the myotubes. Upon exposure to nocodazole, microinjected myotubes exhibited wavy, fluorescent filament bundles around the muscle nuclei. In PtK2 cells, an epithelial cell line, injected desmin formed a filamentous network, which colocalized with the native vimentin intermediate filaments but not with the cytokeratin networks and microtubular arrays. Exposure of the injected cells to nocadazole or acrylamide caused the desmin network to collapse and form a perinuclear cap that was indistinguishable from vimentin caps in the same cells. During mitosis, labeled desmin filaments were excluded from the spindle area, forming a cage around it. The filaments were partitioned into two groups either during anaphase or at the completion of cytokinesis. In the former case, the perispindle desmin filaments appeared to be stretched into two parts by the elongating spindle. In the latter case, a continuous bundle of filaments extended along the length of the spindle and appeared to be pinched in two by the contracting cleavage furrow. In these cells, desmin filaments were present in the midbody where they gradually were removed as the desmin filament network became redistributed throughout the cytoplasm of the spreading daughter cells.
Immunocytochemical studies of spectrin in hamster cardiac tissue
Authors: Messina Dino A. , Lemanski Larry F.
Published: February 2005

The spectrins are a family of cytoskeletal‐membrane proteins that have a wide tissue distribution. In the present study, we employed polyclonal antibodies made against mammalian and avian erythroid spectrins as well as mammalian brain spectrin to assess their presence and distributions in the mammalian heart. Western blot analyses revealed that all three antibodies were specific for a 240,000 molecular weight α‐spectrin subunit found in hamster erythrocyte ghost homogenates, whole hamster heart, and isolated hamster cardiac myofibril homogenates. Spectrin staining was absent from the Triton X‐100‐extracted supernatant fraction of myofibril preparations, suggesting that the protein is linked to the myofibril precipitate after exposure to the detergent. Frozen, unfixed, 2‐μm‐thick; sections of adult, Syrian golden hamster cardiac tissue exhibited strong immunofluorescent staining of intercalated discs and Z‐bands using all three antibodies. In addition, the mammalian erythroid spectrin antibodies showed staining of the sarcolemma, and in cross section, revealed a delicate internal network of staining that appears to surround individual myofibrils. This may be T‐tubule‐associated staining. Myofibrils isolated from cardiac myocytes using Triton X‐100 show positive Z‐band staining using all three antibodies. Double staining with Texas Red‐labeled monoclonal desmin and FITC‐labeled polyclonal spectrin antibodies revealed that both stained the myofibrillar Z‐line regions. These results demonstrate that spectrin is closely associated with the membranes, myofibrils, and intermediate filaments in the mammalian heart.
Establishment of a stable, acetylated microtubule bundle during neuronal commitment
Authors: Falconer Marcia M. , Vielkind Ursula , Brown David L.
Published: February 2005

Two posttranslational modifications of alpha‐tubulin, acetylation and detyrosination, are associated with stable microtubule (MT) populations, including those of neuronal processes. We have used a pluripotent embryonal carcinoma cell line, P19, to investigate changes in MT isotype and stability found in MT arrays during neurogenesis. This cell line has an advantage in that both commitment‐ and differentiation‐related events can be observed. Uncommitted P19 cells have minimal arrays of acetylated and detyrosinated MTs. Following neuronal induction with retinoic acid (RA), indirect immunofluorescence microscopy shows that the first MT modifications occur during commitment and before any morphological change is observed. RA‐induced cells initially polymerize a temporarily enlarged population of MTs. Included in this population is a new array of acetylated MTs arranged in a bundle of parallel MTs. This bundle is colchicine‐stable, although no MT‐associated proteins (MAPs) are detectable using a battery of anti‐MAP antibodies. Observation of MT arrays with patterns that are intermediate between the early bundles and short neurites suggests that the acetylated MT bundle subsequently extends to form a neurite. MAP 2 is first detected at about the time of neurite extension. However, at this early stage of differentiation, MAP 2 is not yet limited to dendritic processes. This report provides the first evidence that the stable MTs of mature neurons may be initiated during neuronal commitment.
Copurification of kinesin polypeptides with microtubule‐stimulated Mg‐ATPase activity and kinetic analysis of enzymatic properties
Authors: Wagner Mark C. , Pfister K. Kevin , Bloom George S. , Brady Scott T.
Published: February 2005

Determination of kinetic properties for kinesin adenosine triphosphatase (ATPase), a proposed motor for transport of membranous organelles, requires adequate amounts of kinesin with a consistent level of enzymatic activity. A purification procedure is detailed that produces approximately 2 mg of kinesin at up to 96% purity from 800 g of bovine brain. This protocol consists of a microtubule affinity step using 5′‐adenylylimidodiphosphate (AMP‐PNP); followed by gel filtration, ion exchange, and hydroxylapatite chromatography; and then sucrose density gradient centrifugation. The microtubule‐activated ATPase activity of kinesin coeluted with kinesin polypeptides throughout the purification. Highly purified kinesin had a Vmax of 0.31 μmol/min/mg in the presence of microtubules, with a Km for ATP of 0.20 mM. The kinetic constants obtained in these studies compare favorably with physiological levels of ATP and microtubules. Variations in buffer conditions for the assay were found to affect ATPase activity significantly. A study of the ability of kinesin to utilize a variety of cation‐ATP complexes indicated that kinesin is a microtubule‐stimulated Mg‐ATPase, but kinesin is able to hydrolyze Ca‐ATP, Mn‐ATP, and Co‐ATP as well as Mg‐ATP in the presence of microtubules. In the absence of microtubules, Ca‐ATP appears to be the best substrate. Studies with several inhibitors of ATPases determined that vanadate inhibited kinesin ATPase at the lowest concentrations of inhibitor, but significant inhibition of the ATPase also occurred with submillimolar concentrations of AMP‐PNP. Other inhibitors of kinesin include N‐ethylmaleimide, adenosine diphosphate (ADP), pyrophosphate, and tripolyphosphate. Further characterization of the kinetic properties of the kinesin ATPase is important for understanding the molecular mechanisms for transport of membranous organelles along microtubules.
Fluorescence microscopic localization of actin in pollen tubes: Comparison of actin antibody and phalloidin staining
Authors: Tang Xiaojing , Lancelle Susan A. , Hepler Peter K.
Published: February 2005

A comparison of actin localization in pollen tubes of Nicotiana has been made using a monoclonal actin antibody and rhodamine‐phalloidin (RP). The monoclonal antiactin, based on Western blotting of pollen tube extract, labels a polypeptide at 45 kD that comigrates with muscle actin. A 51‐kD unknown protein and three bands less than 45 kD, presumed to be proteolytic fragments of actin, are also observed. Structural observaations using this antibody reveal a network of axially oriented strands of microfilaments (MFs). The MFs are distributed throughout the length of the pollen tube except at the very tip, where diffuse staining is usually observed. A similar pattern of MFs is evident after RP staining. When pollen tubes are treated with cytochalasins (CB or CD) cytoplasmic streaming is inhibited, as is tube elongation. Microscopic analysis reveals that the microfilament (MF) pattern is markedly altered; however, the antibody and RP produce different staining patterns. The antibody reveals many MF strands that distribute throughout the tube length and extend into the very tip. In contrast, RP shows mostly a diffuse staining pattern with only a few short clumps of filamentous material. Immunogold labelling of sections of pollen tubes prepared by rapid‐freeze fixation and freeze substitution reveals that actin MF bundles are indeed present after cytochalasin treatment. Our results thus question reports in the literature, based on phalloidin staining, asserting that cytochalasin fragments or destroys actin MFs.
The molecular structure of adrenal medulla kinesin
Authors: Hisanaga Shin‐ichi , Murofushi Hiromu , Okuhara Koji , Sato Reiko , Masuda Yayoi , Sakai Hikoichi , Hirokawa Nobutaka
Published: February 2005

The molecular structure of bovine adrenal kinesin was studied by electron microscopy using the low‐angle rotary shadowing technique. Adrenal kinesin exhibited either a folded or an extended configuration; the ratio of the two is dependent on the salt concentration. Almost all adrenal kinesin molecules were folded in a low‐ionic solution, and the ratio of extended molecules increased to 40–50% in a solution containing 1 M ammonium acetate. Kinesin in the extended configuration displayed a rod‐shaped structure with a mean length of about 80 nm. The morphologies of the ends were different; one end was composed of two globular particles, similar to the two‐headed structure of myosin, while the other end had a more ill‐defined structure, appearing either as a globular particle, an aggregate of two to four small granules, or a frayed, fan‐like structure. The folded kinesin molecule possessed a hinge region in the middle of the rod, at about 32 nm from the neck of the two heads. In our preparations, the majority of adrenal kinesin molecules were folded at physiological salt concentrations. Adrenal kinesin bound to microtubules in the presence of adenylyl imidodiphosphate (AMP‐PNP) also displayed a folded morphology.
Rearrangements of pterinosomes and cytoskeleton accompanying pigment dispersion in goldfish xanthophores
Authors: Palazzo Robert E. , Lynch Thomas J. , Lo Szecheng J. , Taylor John D. , Tchen T. T.
Published: February 2005

The cytoskeleton of goldfish xanthophores contains an abundance of unique dense structures (400 nm in diameter) that are absent in goldfish nonpigment cells and are probably remnants of pterinosomes. No major difference in protein composition between xanthophores and nonpigment cells (without these structures) was found that could account for these structures. In xanthophores, these structures are foci of radiating filaments. The addition or withdrawal of ACTH causes a radical rearrangement of the xanthophore Cytoskeleton accompanying redistribution of carotenoid droplets, namely, the virtual exclusion of these dense bodies with associated filaments from the space occupied by the carotenoid droplet aggregate vs. a relatively even cytoplasmic distribution of these structures when the carotenoid droplets are dispersed. These changes in cytoskeletal morphology are not accompanied by any major changes in the protein or phosphoprotein composition of the cytoskeleton.
Cytoskeletal organization of migrating retinal pigment epithelial cells during wound healing in organ culture
Authors: Hergott Greg J. , Sandig Martin , Kalnins Vitauts I.
Published: February 2005

Retinal pigment epithelial (RPE) cells maintained in organ culture on Bruch's membrane and the associated choroid spread and migrate into a linear wound along the exposed basal lamina. Changes in cell shape, in the organization of microfilaments, and in cell‐cell and cell‐substratum interactions during this time were examined by epifluorescence and transmission electron microscopy. In contrast to cuboidal stationary cells distant from the wound edge, which display well‐developed apical circumferential microfilament bundles (CMBs) associated with zonulae adhaerentes junctions, the migrating RPE cells near the wound edge instead are flat, and, in addition to microfilament bundles near junctions between adjacent cells, display prominent stress fibers. Furthermore, monoclonal antibodies to vinculin labeled regions at the terminal ends of these stress fibers indicating that the RPE cells form focal contacts with the basal lamina at these sites. Electron microscopy of these regions of cell‐substratum interaction confirmed the presence of microfilament bundles that terminate on the cell membrane. Folds present in the basal lamina near these sites suggest that tension is being generated by the microfilaments in the stress fibers as the migrating cells pull on the underlying basal lamina through these adhesion points.
Latrunculins—novel marine macrolides that disrupt microfilament organization and affect cell growth: I. Comparison with cytochalasin D
Authors: Spector Ilan , Shochet Nava R. , Blasberger Dina , Kashman Yoel
Published: February 2005

The latrunculins are architecturally novel marine compounds isolated from the Red Sea sponge Latrunculia magnifica. In vivo, they alter cell shape, disrupt microfilament organization, and inhibit the microfilament‐mediated processes of fertilization and early development. In vitro, latrunculin A was recently found to affect the polymerization of pure actin in a manner consistent with the formation of a 1:1 molar complex with G‐actin. These in vitro effects as well as previous indications that the latrunculins are more potent than the cytochalasins suggest differences in the in vivo mode of action of the two clases of drugs. To elucidate these differences we have compared the short‐ and long‐term effects of latrunculins on cell shape and actin organization to those of cytochalasin D. Exposure of hamster fibroblast NIL8 cells for 1–3 hr to latrunculin A, latrunculin B, and cytochalasin D causes concentration‐dependent changes in cell shape and actin organization. However, the latrunculin‐induced changes were strikingly different from those induced by cytochalasin D. Furthermore, while initial effects were manifest with both latrunculin A and cytochalasin D already at concentrations of about 0.03 μg/ml, latrunculin A caused complete rounding up of all cells at 0.2 μg/ml, whereas with cytochalasin D maximum contraction was reached at concentrations 10–20 times higher. The short‐term effects of latrunculin B were similar to those of latrunculin A although latrunculin B was slightly less potent. All three drugs inhibited cytokinesis in synchronized cells, but their long‐term effects were markedly different. NIL8 cells treated with latrunculin A maintained their altered state for extended periods. In contrast, the effects of cytochalasin D progressed with time in culture, and the latrunculin B‐induced changes were transient in the continued presence of the drug. These transient effects were found to be due to a gradual inactivation of latrunculin B by serum and were used to compare recovery patterns of cell shape and actin organization in two different cell lines. This comparison showed that the transient effects of latrunculin B were fully reversible for the NIL8 cells and not for the mouse neuroblastoma N1E‐115 cells.
Distribution of desmoplakin in normal cultured human keratinocytes and in basal cell carcinoma cells
Authors: Jones Jonathan C. R. , Grelling Kent A.
Published: February 2005

In cultured human keratinocytes (NHEK) maintained in medium containing low levels of Ca2+ (0.04 mM) desmoplakin is a component of certain electron‐dense bodies in the cytoplasm. These bodies are associated with bundles of intermediate filaments. Upon elevation of the level of Ca2+ in the culture medium to 1.2 mM, desmoplakin first appears at sites of cell—cell contact in association with bundles of intermediate filaments. Subsequently, desmoplakin becomes incorporated into desmosomes in a manner comparable to that seen in mouse keratinocytes (Jones and Goldman: Journal of Cell Biology 101:506–517, 1985). NHEK cells maintained for 24 hr at Ca2+ concentrations between 0.04 mM and 0.18 mM were processed for immunofluorescence, immunoelectron, and conventional electron microscopical analysis. In NHEK cells grown at Ca2+ concentrations of 0.11 mM, desmoplakin appears to be localized in electron‐dense bodies associated with intermediate filaments at sites of cell—cell contact in the absence of formed desmosomes. At a Ca2+ concentration of 0.13 mM desmoplakin is arrayed like beads on a “string” of intermediate filaments at areas of cell—cell association. At 0.15 mM, desmosome formation occurs, and desmoplakin is associated with the desmosomal plaque. In basal cell carcinoma cells desmoplakin is not restricted to desmosomes but also occurs in certain electron‐dense bodies morphologically similar to those seen in NHEK maintained in low levels of Ca2+ and during early stages of desmosome assembly. We discuss the possibility of “cycling” of desmoplakin through these bodies in proliferative cells.
What structures, besides adhesions, prevent spread cells from rounding up?
Authors: Zand Martin S. , Albrecht‐Buehler Guenter
Published: February 2005

The outline of cells in sparse cultures consists prediminantly of concave and convex segments; straight segments are rare and ephemeral. The convex segments are areas of active cell expansion. The concave segments are stationary and web‐shaped, similar in profile to the cables of a suspension bridge. In 3T3 fibroblasts, we have found a single microfilament bundle following the outline of every webbed edge and have called it the actin edge‐bundle (AEB). While the AEB is composed predominantly of actin, α‐actinin and myosin are also present. In contrast to normal stress fibers, AEBs are more resistant to several treatments that depolymerize F‐actin. Once an AEB disassembles, however, the webbed edge collapses and retracts, suggesting that the actin edge‐bundle is a specialized cytoskeletal structure that supports the webbed edges of interphase 3T3 fibroblasts. The stability of AEBs is independent of microtubules. We suggest that the microfilament bundles that frequently line the lateral contacts between epithelial cells in vivo may be related to the actin edge‐bundle.
Rapid kinetochore movements in Mesostoma ehrenbergii spermatocytes: Action of antagonistic chromosome fibres
Authors: Fuge Harald
Published: February 2005

Chromosome movements in Mesostoma ehrenbergii spermatocytes were studied using conventional video light microscopy. Kinetochore regions of the three bipolarly oriented bivalents displayed periodic back and forth movements directed to both poles at metaphase I, leading to periodic lenght changes of the bivalents. Velocity was 8–10 μm/min (maximum 17 μ/min), about one order of magnitude higher than the normal meiotic or mitotic chromosome movements of other species. One cycle of movement lasted for about 100 seconds. The movement of kinetochore regions implies that the antagonistic chromosome fibres periodically grow (assemble) and shorten (disassemble) at comparable rates. Poleward movements must be caused by forces generated in disassembling fibres, whereas movements away from the poles, accompanied by fibre growth, are probably brought about by the internal elastic force of the chromosomes. Antagonistic fibres of a bivalent can operate in or out of phase. The movements of the three kinetochore regions are coordinated insofar as growing and shortening fibres coexist in a half‐spindle at almost any time [Fuge, H. (1987): European Journal of Cell Biology 44:294–298]. These observations are discussed in terms of microtubule dynamics.
Dynamics of the endoplasmic reticulum in living non‐muscle and muscle cells
Authors: Sanger Jean M. , Dome Jeffrey S. , Mittal Balraj , Somlyo Avril V. , Sanger Joseph W.
Published: February 2005

The dynamic changes of the endoplasmic reticulum (ER) in interphase and mitotic cells was detected by the vital fluorescent dye 3,3′‐dihexyloxacarbocyanine iodide. Two types of arrays characterize the continuous ER system in the non‐muscle PtK2 cell: (1) a lacy network of irregular polygons and (2) long strands of ER that are found aligned along stress fibers. In cross‐striated myotubes there was a periodic localization of fluorescence over each I‐band corresponding to the positions of the terminal cisternae of the sarcoplasmic reticulum (SR). In contrast to the arrangement in muscle cells, the aligment of the long strands of ER along stress fibers showed no strict periodicity that could be correlated with the sarcomeric units of the stress fibers. The ER and SR arrays seen in living cells were also detected in fixed cells stained with antibodies directed against proteins of the endoplasmic reticulum and sarcoplasmic reticulum, respectively. Observations of vitally stained PtK2 cells at 1 to 2 minute intervals using low light level video cameras and image processing techniques enabled us to see the polygonal ER units form and undergo changes in their shapes. During cell division, the ER, rhodamine 123‐stained mitochondria, and phagocytosed fluorescent beads were excluded from the mitotic spindle while soluble proteins were not. No obvious concentration or alignment of membranes could be found associated with the contractile proteins in the cleavage furrow. After completion of cell division there was a redeployment of the ER network in each daughter cell.
Synthesis of mammalian profilin in Escherichia coli and Its characterization
Authors: Babcock Gary , Rubenstein Peter A.
Published: February 2005

Profilin is a G‐actin binding protein that may have a role in controlling the ratio of G/F actin within the cells To devise a way for obtaining large amounts of mammalian profilin in an active state, we transfected Escherichia coli with a plasmid containing a full‐length rat spleen profilin cDNA adjacent to a promoter inducible by isopropyl thiogalactoside (IPTG). Upon induction, they synthesized a new protein of 15,000 MW constituting approximately 5% of the total cell protein. This protein bound to poly‐L‐proline Sepharose and could be eluted with 7 M urea, behavior similar to that exhibited by authentic profilin. The protein could be released from the bacteria in soluble form following sonication, and the profilin could then be purified to homogeneity following chromatography on Sephadex G‐75 and DEAE A‐50 Sephadex. The protein began with an unblocked Ala, indicating that the initiating formyl and methionine residues had been removed. The dissociation of the recombinant profilin from chicken skeletal muscle actin was characterized by a Kd of approximately 2 μM based on gel filtration analysis and actin polymerization assays. These results show that purified active mammalian profilin can be made conveniently in large quantities. This study also demonstrates the feasibility of using bacterially synthesized profilin in structure‐function studies involving mutant profilins altered by site‐directed mutagenesis.
Acetylated α‐tubulin in spermatogenic cells of the crane fly Nephrotoma suturalis: Kinetochore microtubules are selectively acetylated
Authors: Wilson P. J. , Forer A.
Published: February 2005

We studied the distribution of acetylated α‐tubulin in the microtubules of spermatogenic cells from the crane fly Nephrotoma suturalis (Loew) using a mono‐clonal antibody specific for acetylated α‐tubulin (6‐11B‐1). We found that cells in all stages of spermatogenesis contained acetylated microtubules including primary spermatocytes, meiotic cells, spermatids, and sperm. A subset of the acety‐lated microtubules (those in midbodies and flagella) were resistant to cold depolymerization. Newly polymerized microtubules in nondividing cells were not acetylated for up to 15 min. indicating that acetylation lagged behind polymerization. In spindles, newly polymerized microtubules were acetylated after 5 min. Antibodies to acetylated α‐tubulin selectively stained chromosome‐to‐pole fibers in dividing cells, but the staining appeared to decrease and taper of at the kinetochores. This observation supports the hypothesis that tubulin subunits add at the kinetochore in metaphase and that acetylation occurs subsequent to addition. Further, this taper may be useful as a marker in anaphase, to distinguish between different hypotheses of chromosome motion.
Talin dynamics in living microinjected nonmuscle cells
Authors: Hock Rick S. , Sanger Jean M. , Sanger Joseph W.
Published: February 2005

To investigate the role of talin in the anchoring of actin‐containing stress fibers to the cell membrane of nonmuscle cells, a fluorescent analog of the adhesion plaque protein talin was developed, characterized, and microinjected into living cells. Purified chicken gizzard talin was covalently labeled with the fluorescent dye lissamine rhodamine B sulfonyl chloride. The fluorescently labeled protein was then chromatographed on Sephadex G‐25 and DEAE‐cellulose in order to remove free dye and denatured protein. The fluorescent talin was able to bind purified vinculin and was localized in adhesion plaques, membrane ruffles, microspikes, and polygonal networks in acetone‐permeabilized nonmuscle cells. In cells that were double‐stained with fluorescent talin and an affinity‐purified anti‐talin an‐tibody, a one‐to‐one correspondence of adhesion plaque staining was seen. Living epithelial cells (PtK2) were microinjected during interphase with fluorescent talin. Computer‐enhanced video microscopy was used to document adhesion plaque dynamics such as (1) changes in plaque shape, (2) alterations in plaque positions, and (3) the appearance, growth, and dissolution of plaques. In cells that were followed during mitosis, the adhesion plaques disappeared during cell rounding and then subsequently reappeared upon spreading of the two daughter cells. Treatment of microinjected cells with DMSO in order to disassemble stress fibers resulted in an altered localization of the fluorescent talin. Upon recovery of the cell from the drug, the talin was visualized in its characteristic submembraneous position. These results are the first to document the role and distribution of talin in dynamic processes occurring in living microinjected nonmuscle cells.
Splitting the ciliary axoneme: Implications for a “Switch‐Point” model of dynein arm activity in ciliary motion
Authors: Satir Peter , Matsuoka Tatsuomi
Published: February 2005

In the presence of specific inhibitors of beat, 20 μM VO43− or pCa 4, mussel gill lateral (L) cilia can be arrested in two positions—“hands down” or “hands up”—at opposite ends of the stroke cycle. Cilia move to these positions by doublet microtubule sliding. Axonemes of arrested cilia, still tethered to the cell, are intact after demembranation and protease treatment. When reactivated by 4 mM ATP with inhibitors present, about 40% split apart. Splits are not random but occur preferentially between different specific doublets in the two opposite arrest positions. Several different related patterns of splitting are observed; for every pattern in “hands down” axonemes, there is a corresponding complementary split pattern in “hands up” axonemes. In some split patterns two doublets remain firmly attached to the central pair; these also differ depending on axonemal position. Although some of the patterns seen may be artifactual or difficult to explain, the complementary splitting patterns are predictable with simple assumptions by a “switch point” hypothesis of ciliary activity where, during each recovery stroke, doublets 6–8 have active dynein arms, while during each effective stroke, arms on doublets 1–4 become active, and arms 6–8 are turned off. Because of a difference between the patterns seen and the predictions, the status of the arms on doublet 9 is unresolved. The patterns also suggest that a spokecentral sheath attachment cycle may correlate with switching of arm activity during the generation of an asymmetric beat.
Relationship between tektins and intermediate filament proteins: An immunological study
Authors: Steffen Walter , Linck Richard W.
Published: February 2005

Affinity‐purified antibodies raised against three flagellar tektins (tektin A, B, and C) from each of two sea urchin species (Lytechinus pictus and Strongylocentrotus purpuratus) were used to study the immunological relationship between tektins and intermediate filament proteins. By immunofluorescence microscopy, several antitektins revealed a staining of intermediate filament‐like arrays in three vertebrate cell lines tested. Immunoelectron microscopy substantiated the cross reaction of antitektins with intermediate filaments. When the cells were treated with cytochalasin B, the arrangement of the filaments recognized by anti‐(Lp)‐tektin B was altered; the alteration observed is typical for keratin filaments. By immunoblot, it was found that anti‐(Lp)‐tektin B cross reacted with two isoforms or different proteins of ∼︁54 kD with pIs of 6.1 and 6.2 in human carcinoma epithelia (HeLa) cells and with two isoforms or different proteins of ∼︁55 kD with pIs of 6.1 and 6.3 in pig kidney epithelia (LLC‐PK1) cells. Furthermore, when antitektin antibodies were affinity purified with the 54 kD HeLa keratin, these keratin‐specific antibodies again restained the original tektins on immunoblots. From these observations, it can be concluded that tektins and keratins are to a certain extent immunologically related. To determine the degree of the immunological relationship, tektin filaments and purified intermediate filaments from HeLa cells were cleaved with α‐chymotrypsin and examined by quantitative immunoblot analysis. On immunoblots of digested tektins from L. pictus, anti‐(Lp)‐tektin B recognized several cleavage products in the range of 20 kD to 46 kD. However, when immunoblots of digested intermediate filaments from HeLa cells were probed, the cross reaction of anti‐(Lp)‐tektin B with HeLa keratins was eliminated by more than 98% within 2 min, suggesting that tektins have epitopes in common with the end domains of certain keratins.
Morphology of fibroblasts in collagen gels: A study using 400 keV electron microscopy and computer graphics
Authors: Heath Julian P. , Peachey Lee D.
Published: February 2005

We have used 400 kilovoit intermediate voltage electron microscopy (IVEM) to examine thick sections of fibroblasts cultured in collagen gels. In these 3D collagen lattices, the long, narrow pseudopodial extensions that extend out and make contact with the collagen matrix exhibit a complex topography not seen in the processes put out by cells moving on planar substrata. For this reason, sections 1 to 2 μm thick that enclose a whole cell process are more informative of the overall morphology of the interaction between cells and the collagen than are thin sections. To aid the discrimination of topography of cell processes in stereo views of micrographs, some cells were labeled with antibodies and protein A‐colloidal gold conjugates. The gold particles provided clear 3D reference points for computeraided reconstructions of membrane topography from tilt series of IVEM images. Our results confirm that cells that move through collagen lattices lack the wellspread morphology of their counterparts moving on glass. They are generally rather spindly with several long branching anterior pseudopodia. We found that the cell bodies and major pseudopodial processes were cylindrical, as one might expect of cells in a 3D environment, but at the leading edge of advancing pseudopodia there are small flat extensions similar to those seen in cells on glass. This similarity suggests that the lamellipodium is a basic type ofprotrusive structure used by fibroblasts during locomotion on all types of substratum. The flattened shape of lamellipodia may be part of the mechanism by which cells sense the orientation of fibrillar extracellular matrices during embryonic morphogenesis.
Isolation of a subpellicular microtubule protein from Trypanosoma Brucei that mediates crosslinking of microtubules
Authors: Balaban N. , Waithaka H. K. , Njogu A. R. , Goldman R.
Published: February 2005

The cell body of Trypanosomatidae is enclosed in densely packed, crosslinked, subpellicular microtubules closely underlying the plasma membrane. We isolated the subpellicular microtubules from bloodstream Trypanosoma brucei parasites by use of a zwitterion detergent. These cold stable structures were solubilized by a high ionic strength salt solution, and the soluble proteins that contained tubulin along with several other proteins were further fractionated by Mono S cation exchange column chromatography. Two distinct peaks were eluted containing one protein each, which had an apparent molecular weight of 52 kDa and 53 kDa. (Mr was determined by SDS‐gel electrophoresis.) Only the 52 kDa protein showed specific tubulin binding properties, which were demonstrated by exposure of nitrocellulose‐bound trypanosome proteins to brain tubulin. When this protein was added to brain tubulin in the presence of taxol and GTP, microtubule bundles were formed with regular crosslinks between the parallel closely packed microtubules. The crosslinks were about 7.2 nm apart (center to center). Under the same conditions, but with the 53 kDA protein or without trypanosome derived proteins, brain tubulin polymerized to single microtubles. It is thus suggested that the unique structural organization of the subpellicular microtubules is dictated by specific parasite proteins and is not an inherent property of the polymerizing tubulin. The in vitro reconstituted microtubule bundles are strikingly similar to the subpellicular microtubule network of the parasite.
Association of ribosomes with in vitro assembled microtubules
Authors: Suprenant Kathy A. , Tempero Libeth B. , Hammer Lorraine E.
Published: February 2005

Microtubules were purified from unfertilized eggs of the sea urchins Arbacia punctulata, Lytechinus pictus, Lytechinus variegatus, and Strongylocentrotus purpuratus. Numerous densely stained particles (24 × 26 nm) are associated with microtubules isolated from each of these sea urchins. The most striking aspect of this structure is an extended, slightly curved arm that appears to attach the particles to the microtubule. Morphologically similar particles are associated with microtubules of the isolated first cleavage mitotic apparatus. The particles are attached to the microtubules by ionic interactions and contain large amounts of extractable RNA. Based upon their size and density, RNA and protein composition, and sedimentation in sucrose gradients, the microtubule‐associated particles are identified as ribosomes.
Purification of anterogin, a protein factor necessary for the dispersion of carotenoid droplets in permeabilized xanthophores of goldfish
Authors: Zeng Zhao‐Chun , Taylor John D. , Tchen T. T.
Published: February 2005

We reported previously that the dispersion of carotenoid droplets in permeabilized xanthophores requires cAMP, ATP, and a cytosolic factor present in several secretory tissues as well as in xanthophores. We have now purified this factor from beef liver to apparent/near homogeneity. It appears to be a heterodimer with Mr ∼125,000. The purified factor has little or no ATPase activity, with or without the presence of actin. Nor does it stimulate the ATPase activity of carotenoid droplets. Its exact function in carotenoid droplet dispersion is thus unclear. Since dispersion of carotenoid droplets is an anterograde translocation, we propose the name anterogin for this protein. We also report that yeast cytosol has anterogin activity.