bluuebunny/crossref_metadata_2025 huggingface dataset:
Flow birefringence of microtubules and its relation to birefringence measurements in cells
Authors: Hard Robert , Allen Robert D.
Published: February 2005

Understanding the molecular basis of mitotic movements in living cells will require correlative experiments on intact cells, cell models, purified tubulin, and perhaps other biopolymers. Birefringence is one assay that is useful in all of these experimental situations. Heretofore, studies of birefringence changes during mitosis have lacked a quantitiative basis for interpretation in terms of microtubule number and packing density. One of the aims of this work was to establish that relationship.

Purified calf brain tubulin was polymerized to equilibrium and oriented in the hydrodynamic field of a microcapillary flow birefringence apparatus. The relationship between birefringence and microtubule packing density was determined by a combination of optical, electron microscopic, and biochemical methods. The data correlate surprisingly well with those obtained by others from in vitro measurements on isolated mitotic spindles. Using the flow birefringence data, the sensitivity of polarizing microscopes for detecting microtubules was examined and found to depend on microtubule packing density, object thickness, and instrumental factors that limit both the detection and measurement of weakly birefringent objects. Because of the dependence of measurement sensitivity on object thickness, a method of measuring the thickness of microtubule bundles using the dispersion of birefringence was developed. This method is capable of measuring thickness to within two or three Airy diffraction units and does not require any assumptions regarding object symmetry.
Bending patterns of Chlamydomonas flagella: II. Calcium effects on reactivated Chlamydomonas flagella
Authors: Omoto C. K. , Brokaw C. J.
Published: February 2005

Ca2+ has profound effects on the movement of cilia and eukaryotic flagella, including those of Chlamydomonas. Two clear changes seen in Chlamydomonas flagella with changes in Ca2+ are beat frequency and symmetry. Photographic and computer assisted analysis of flagellar bending patterns on a uniflagellate mutant of Chlamydomonas have been used to examine details of the effects of Ca2+ on the movement of ATP‐reactivated, demembranated flagella. In addition to the forward mode bending pattern seen at low Ca2+ concentrations (10−9 M), which has a frequency of about 50 Hz and the reverse mode bending pattern seen at high Ca2+ concentrations (10−4 M) with a frequency around 70 Hz, we carefully examined bending patterns in the intermediate Ca2+ concentration range of 1–6.5 × 10−6 M. In this intermediate range, the bending patterns have significantly reduced asymmetry and slightly increased frequency, compared to the motility observed at low Ca2+ concentrations. These observations indicate that changes in these two parameters of motion do not occur in parallel and suggest that the effects of Ca2+ may be a multicomponent process. Physiologically, these changes in the beat pattern at intermediate Ca2+ may signal either (1) the beginning stages of transition to the symmetrical, high‐frequency beating seen at high Ca2+, or (2) a more normal forward mode motility for the trans flagellum as suggested by Kamiya and Witman [1984]. No large amplitude bending patterns associated with transitions between forward and reverse mode beating in intact cells were seen at the intermediate Ca2+ concentrations.
Video microscopy of fast axonal transport in extruded axoplasm: A new model for study of molecular mechanisms
Authors: Brady Scott T. , Lasek Raymond J. , Allen Robert D.
Published: February 2005

The development of AVEC‐DIC microscopy and the application of this method to the study of fast axonal transport in isolated axoplasm extruded from the giant axon of the squid Loligo pealei provides a new paradigm for analyzing the intracellular transport of membranous organelles. The size of the axon, the number of transported particles, and the absence of permeability barriers like the plasma membrane in this preparation permit many experiments that are difficult or impossible to perform using other model systems. The use and features of this preparation are described in detail and a number of properties are evaluated for the first time. The process of extrusion is characterized. Particle movement is evaluated both in the interior of extruded axoplasm and along individual fibrils that extend from the periphery of perfused axoplasm. The role of divalent cations, particularly Ca2+, and the effects of elevated Ca2+ on axoplasmic organization and transport are analyzed. A series of pharmacological agents and polypeptides that alter cytoskeletal organization are used to examine the role of microfilaments and microtubules in fast transport. Finally, the effects of depleting ATP and of adding ATP analogues are discussed. The extruded axoplasm preparation is shown to be an invaluable model system for biochemical and pharmacological analyses of the molecular mechanisms of intracellular transport.
Actin assembly and filament cross‐linking in the presence of TW 260/240, the tissue‐specific spectrin of the chicken intestinal brush border
Authors: Fishkind Douglas J. , Mooseker Mark S. , Bonder Edward M.
Published: February 2005

TW 260/240 is a tissue‐specific spectrin found in the terminal web region of the chicken intestinal bruish border. We have examined the effects of TW 260/240 on assembly rates and critical concentrations (Co's) for monomer addition at the barbed and pointed ends of the actin filament. For these studies, acrosomal processes (AP) from Limulus sperm were used as nuclei for actin assembly. Under conditions which favor the interaction of TW 260/240 for actin (20–75 mM KCl, 2 mM Mg++) no effect on either elongation rates or Co's at either end of the actin filament was observed in the presence of this spectrinlike protein. The Limulus AP nucleation assay also allowed visualization of the kinetics of filament binding and cross‐linking by TW 260/240. Ultrastructural analysis of TW 260/240 binding to actin filaments at their growing ends indicates that TW 260/240 tetramers bind laterally to the filament. Finally, evidence is presented that indicates that filaments cross‐linked by TW 260/240 are stabilized against shear‐dependent breakage.
Live and reactivated motility in the 9 + 0 flagellum of Anguilla sperm
Authors: Gibbons Barbara H. , Baccetti B. , Gibbons I. R.
Published: February 2005

The sperm flagella of the eel, Anguilla anguilla, are capable of vigorous motion in spite of having an axoneme with reduced structure that lacks the outer dynein arms, radial spokes and spoke heads, the two central tubules and the central tubule projections that are all part of the standard “9+2” axoneme. These sperm progress forward rapidly as a result of the propagation of helicoidal waves distally along the flagellum. Their flagellar beat frequencies are high, 93 Hz at 21°C, and they roll at a frequency of about 19 Hz. Eel sperm could be demembranated with Nonidet P‐40 and reactivated with MgATP2− in 0.22 M K acetate at pH 8.1. The reactivated motility closely resembles that of the live sperm, with a beat frequency of 69 Hz, but the demembranated flagella are unusually fragile, and commonly disintegrate by a combination of splitting, coiling, and sliding within a few minutes. Little reactivation is obtained if acetate is replaced by Cl− in the reactivating medium. The Michaelis constant for beat frequency (0.2 mM) is similar to that obtained for several “9+2” flagella. These sperm, however, appear to lack the mechanism by which Ca2+ regulates waveform. Our results indicate that eel sperm flagella, which at rest are straight, are induced to bend helicoidally by ATP, as the result of sliding between tubules that is blocked at both the base and tip of the organelle. The flagellar waveform consists of a series of planar bends separated by short regions of right‐handed twist, which give it an overall left‐handed helicoidal form.
Newt lung ciliated cell models: Effect of MgATP on beat frequency and waveforms
Authors: Weaver Alice , Hard Robert
Published: February 2005

Highly coupled newt lung ciliated cell models were used to study the effects of MgATP concentration on ciliary beat frequency and waveform. Models were prepared from ciliated lung cells of the newt Taricha granulosa by trypsin dissociation of the epithelium, demembranation with Triton X‐100, and reactivation with MgATP, as described previously [Weaver and Hard, 1985]. Beat frequencies were measured stroboscopically. Ciliary waveforms of reactivated models and intact mucociliary epithelial sheets were determined by single frame analysis of high‐speed movies. Waveform parameters calculated included the durations of the effective and recovery strokes, the angular velocities of the ciliary base and tip, the position of the bend along the ciliary shaft during the recovery stroke, the velocity of recovery stroke bend propagation, and the ratio of the duration of recovery stroke bend propagation to the duration of the recovery stroke itself. We found that beat frequency varied biphasically in response to MgATP at 21°C, as shown previously for isolated, individual, newt lung axonemes. Apparent Fmax (maximum beat frequency) and Km values of 25 Hz and 0.14 mM, and 35 Hz and 0.47 mM, respectively, were obtained for each linear segment of the biphasic double reciprocal plot. Demembranation did not alter either ciliary waveform or the pattern of coordination. In this system, metachrony is antilaeoplectic and ciliary waveform appears to be regulated independent of beat frequency.
A Method for quantifying F‐Actin in chemotactic peptide activated neutrophils: Study of the effect of tBOC peptide
Authors: Howard Thomas H. , Oresajo C. O.
Published: February 2005

The studies presented here characterize a simple, quantitative NBDphallacidin extraction assay for determining the F‐actin content of fMLP‐activated neutrophils. The NBDphallacidin extraction assay is based upon the specificity of NBDphallacidin binding to F‐actin and the solubility of NBDphallacidin in methanol. Cells are fixed, permeabilized, and stained with NBDphallacidin; the cells are then pelleted, the bound NBDphallacidin is extracted into methanol, and the RFI (excite 465; emit 535) of the solution is determined. Binding of NBDphallacidin to neutrophils is saturable and 90% of bound NBDphallacidin is displaced by nonfluorescent phalloidin. The extraction of bound NBDphallacidin into methanol is complete and the excitation/emission characteristics of NBDphallacidin are not altered by extraction. The assay is relatively inexpensive, applicable to the study of cells in suspension or on substratum, allows kinetic studies with 5–10s time resolution, and is not affected by the shape of the cell or the distribution of the probe. We used the NBDphallacidin extraction assay to study the kinetics of fMLP‐induced change in the F‐actin content of neutrophils and the effect of tBOC peptide, an inhibitor of fMLP binding, on these changes. The extraction assay reveals a rapid, sequential fMLP‐induced increase followed by a decrease in F‐actin content. The tBOC peptide inhibits fMLP‐induced actin polymerization. Addition of tBOC during fMLP‐induced polymerization or at times when F‐actin content is maximal enhances F‐actin depolymerization. The rate of F‐actin depolymerization is ⩾ fourfold faster in the presence than in the absence of tBOC. The results show that (1) The NBDphallacidin extraction assay is useful for studying the kinetics of change in F‐actin content of nonmuscle cells; (2) fMLP receptor occupancy is required for fMLP‐dependent polymerization but not depolymerization; and (3) both the actin polymerizing and depolymerizing processes are active in the cell within 5 s after fMLP stimulation. Implications of these observations for understanding the observed increase and, then, decrease in F‐actin content of fMLP‐activated cells are discussed.
A new model of reticulopodial motility and shape: Evidence for a microtubule‐based motor and an actin skeleton
Authors: Travis Jeffrey L. , Bowser Samuel S.
Published: February 2005

Cytoskeletal inhibitors were used as probes to test the involvement of microtubules and actin microfilaments in the development, motility, and shape maintenance of the pseudopodial networks (i e, reticulopodia) of the foraminifers Allogromia sp strain NF and Allogromia laticcllaris. Agents that disassemble cytoplasmic microtubules (cold, colchicine, and nocodazole) arrest all movement but have variable effects on reticulopodial shape. Electron microscopy reveals a granulofibrillar matrix but few, if any, microtubules in these motility‐arrested reticulopods. Allogromiids treated with cytochalasin B or D lose substrate adhesion and undergo dramatic changes in shape and motile behavior, highlighted by the coalescence of reticulopodial cytoplasm into irregularly shaped bodies with chaotic motility. Serial semithick sections of such preparations, viewed by high‐voltage electron microscopy, document a striking rearrangement of microtubules within these cytochalasin‐induced bodies. All aspects of cytochalasin‐altered motility are completely inhibited by colchicine. Actin is present in reticulopodia, as determined by staining with rhodamine‐phalloidin; this staining is not observed in cytochalasin‐treated organisms. These data provide compelling evidence that microtubules are required for reticulopodial motility. An actin‐based cytoskeleton is thought to play a role in maintaining shape, mediating pseudopod/substrate adhesion, and coordinating the various microtubule‐dependent processes.
Isolation of cilia from porcine tracheal epithelium and extraction of dynein arms
Authors: Hastie Annette T. , Dicker David T. , Hingley Susan T. , Kueppers Friedrich , Higgins Michael L. , Weinbaurn George
Published: February 2005

Milligram amounts of mammalian ciliary axonemes were isolated from porcine tracheas. These were reactivated upon addition of ATP, indicating intact functional capability with a mean beat frequency at 37°C of 8.2 Hz. Electron microscopy showed typical ultrastructure of the isolated demembranated axonemes. Electrophoresis into polyacrylamide gradient gels containing sodium dodecyl sulfate revealed reproducible protein profiles from ten different tracheal preparations. Four major protein bands were observed in the 300–330 K molecular weight region, as well as tubulin at 51–54K. Extraction of the isolated tracheal axonemes with 0.6M KCl removed the outer dynein arms seen in electron microscopic cross‐section of axonemes, preferentially solubilized two of the high molecular weight proteins at 320 and 330 K, and resulted in a three‐ to four‐fold increase in ATPase specific activity. Sedimentation of the dialyzed salt extract on a 5–30% sucrose density gradient and subsequent fractionation yielded two peaks of ATPase activity. The faster migrating, 19S major ATPase peak correlated with the 320 and 330 K proteins, and two other proteins at 81 and 67 K. The slower sedimenting, 12S minor ATPase peak corresponded to a 308 K protein and two smaller proteins at 33 and 48 K. Thus, the outer dynein arm of tracheal cilia appeared to be associated with at least two high molecular weight proteins. These results demonstrate that adequate quantities of functionally intact axonemes can be reproducibly isolated from porcine tracheas, allowing further fractionation and analysis of mammalian cilia.
Abnormal distribution of the periaxonemal structures in a human sperm flagellar dyskinesia
Authors: Serres Catherine , Feneux Danielle , Jouannet Pierre
Published: February 2005

The spermatozoa from four infertile patients showing a flagellar dyskinesia due to abnormal flagellar wave development have been studied by light and transmission electron microscopy (TEM) for flagellar morphology. No axonemal anomalies were found but modification of the periaxonemal structures was observed. The results of a stereological analysis revealed abnormal extension of the individual dense fibres along the axoneme in the four cases as compared with a control group. The order of termination of those structures was therefore altered. However, the overall fibre extension was the same in both groups (ie, 60% of the principal piece). The number and the location of the longitudinal columns were also modified, the predominant anomaly being the presence of a single column. The possible influence of those structural anomalies on the pattern of sperm movement is discussed. Our observations seem to agree with a previous hypothesis of the literature, that the dense fibres might play a role in flagellar flexibility. More particularly, we suggest that the abnormal extension of dense fibres No. 2, 3, and 4 and the symmetric distribution of the dense fibres on both sides of the plane of beating may alter the flagellar curvature amplitude and the cell rotation frequency.
Parameters of the ciliary cycle under membrane voltage control
Authors: Machemer H. , Sugino K.
Published: February 2005

Axial views of depolarization‐ and hyperpolarization‐dependent activation of the frontal cirri of Stylonychia were cinematically recorded at high rate (250 frames/s) under voltage‐clamp. Images of a cirrus performing the cycle were processed by using computer assistance. In responding to the polarity and amplitude of the voltage signal, a cirrus inclines proximally with a particular angle and orientation. The ciliary cycle–always counterclockwise–is superimposing upon steady inclination. Correction for inclination allowed the assessment of the directional change rate and, after inclusion of the amplitude data, the determination of the ciliary angular velocity during the cycle. The method serves to isolate a new ciliary parameter: inclination, and to register precisely parameters of the cycle which may be meaningful for the understanding of the sliding mechanism.
Towards a new classification of intracellular particle movements based on quantitative analyses
Authors: Weiss Dieter G. , Keller Franz , Gulden Josef , Maile Willi
Published: February 2005

A survey study of organelle movements in a variety of cell types of plant and animal origin was made with the aid of video‐enhanced contrast, differential interference contrast (AVEC‐DIC) microscopy followed by fine analysis of the motile behavior of the individual organelles. We found that there exists besides Brownian motion a wide spectrum of active motions in cells, i.e. motion that is directionally biased through the expenditure of metabolic energy. The types of active motion seen range from a continuous motion (sometimes appearing as streaming) in plant cells and neurons to various types of less ordered and less well directed motion. We did not see any clear‐cut qualitative difference between plant and animal cells or between systems presumed to be actin‐ and microtubule‐based. A preliminary classification of the types of active motion is presented. The ongoing research activities, which aim at a more precise definition of the different types of motion by a set of quantitative parameters, are described, and the progress made so far is reported.
Polarized cytoplasmic movement and inhibition of saltations induced by calcium‐mediated effects of microbeams in fungal hyphae
Authors: McKerracher L. J. , Heath I. B.
Published: February 2005

We have investigated the mechanisms that hyphae of the fungus Basidiobolus magnus use to accomplish bulk movement of their cytoplasm and saltatory organelle movements. When cells were irradiated with an ultraviolet microbeam, cytoplasmic contraction occurred. The posterior cytoplasm (toward the septum) always moved forward toward the irradiated area, whereas anterior cytoplasm (between the cell tip and target) never contracted back toward the site of irradiation. Thus, there is an intrinsic polarity in the cytoplasm. Irradiations also arrested saltatory movements. The effects of irradiation on both saltations and cytoplasmic movement appear to be mediated by Ca++. Chelating exogenous Ca++ before irradiation eliminated contractions and prevented the inhibition of saltations. Furthermore, the effects of irradiation could be duplicated by using the Ca++ ionophore A23187. We relate the present results to our previous report on the effects of irradiation on the cytoskeleton [McKerracher and Heath, 1986]. We conclude that two separate cytoskeletal networks exist in fungal cells, and that an actin‐containing network generates bulk cytoplasmic movement.
Structural evidence for contractile units in the giant smooth muscle cell of Beröe
Authors: Hernandez‐Nicaise M. L. , Nicaise G.
Published: February 2005

Bundles of giant smooth muscle fibers of the ctenophore Beröe have been stretched up to four times their initial length and then examined by transmission electron microscopy. Stretching induces the segregation of actin and thick (myosinlike) filaments into separate domains. The thick filaments domains are made of 20–30 filaments, with a regular spacing identical to that of nonstretched fibers. A moderate stretching permits the visualization of microtendons associating actin filament bundles to hyaluronidase‐resistant condensations of the extracellular matrix. It is deduced from these observations that Beröe giant smooth muscle fibers possess myofibrils which attach at both ends upon the sarcolemmal membrane. Each myofibril is probably made of two long actin filament bundles (of approximately 150 filaments) and short (2–3 μm) myosinlike bundles (of approximately 30 filaments).
The mechanical behavior of doublet microtubules simulated by helical models
Authors: Jarosch Robert
Published: February 2005

The structural and mechanical properties of the helix‐shaped isolated doublet microtubules described by Costello [Biol. Bull. 145:279–291, 1973], Zobel [J. Cell Biol. 59:573–94, 1973], and Miki‐Noumura and Kamiya [Exp. Cell Res. 97:451–53, 1976, J. Cell Biol 81:355–60, 1979] are simulated by a left‐handed superhelix model that consists of two intertwined springlike helices with a slight difference in their pitches. It is shown by combinations of two and more superhelices of this kind that the straight shape of the doublets in the axoneme is the consequence of a position‐dependent mechanical coil‐coil interaction between interconnected doublets whose curvatures bend against one another. A counterclockwise torsion changes the sense of the superhelix from the left‐handed form with a smaller pitch (L2) to the right‐handed form with a larger pitch (R3). The coil‐coil interaction of L helices with R helices results in uniplanar, meanderlike shapes or in flattened helices. The one‐sided distribution of L and R doublets as described for sperm axonemes by Costello [Biol. Bull. 145:279–91, 1973] may therefore be responsible for the uniplanar. meanderlike shape of these flagella.
Independence of centriole formation and initiation of DNA synthesis in Chinese hamster ovary cells
Authors: Kuriyama Ryoko , Dasgupta Santanu , Borisy Gary G.
Published: February 2005

The relationship between centriole formation and DNA synthesis was investigated by examining the effect of taxol on the centriole cycle and the initiation of DNA synthesis in synchronized cells. The centriole cycle was monitored by electron microscopy of whole‐mount preparations [Kuriyama and Borisy, J. Cell Biol., 1981, 91:814–821]. A short daughter centriole appeared in perpendicular orientation to each parent during late G1 or early S and elongated slowly during S to G2. Addition of 5–20 μg/ml taxol to a synchronous population of cells in S phase did not inhibit centriole elongation; rather, elongation was accelerated. In contrast, when taxol was added to M phase or early G1 cells, centriole duplication was completely inhibited. The taxol block was reversible since nucleation and elongation of centrioles resumed as soon as the drug was removed. Cells exposed to taxol progressed through the cell cycle and became blocked in mitosis, as indicated by an increase in the mitotic index, but eventually the mitotic arrest was overcome, resulting in formation of multinucleated cells. A peak in mitotic index was seen in the following generation, indicating that chromosomes duplicated in the presence of taxol. Incorporation of 3H‐thymidine followed by autoradiography confirmed that DNA synthesis was initiated in the presence of taxol even though formation of daughter centrioles was inhibited. It seems, therefore, that centriole duplication is not a prerequisite for entry into S phase. Since DNA synthesis has already been demonstrated not to be necessary for centriole duplication, these two events, normally coordinated in time, appear to be independent of each other.
Structure and composition of the cytoskeleton of nucleated erythrocytes: III. Organization of the cytoskeleton of Bufo marinus erythrocytes as revealed by freeze‐dried platinum‐carbon replicas and immunofluorescence microscopy
Authors: Centonze Victoria E. , Ruben George C. , Sloboda Roger D.
Published: February 2005

Platinum‐carbon (Pt‐C) replicas of freeze‐dried erythrocyte cytoskeletons of the toad, Bufo marinus, were prepared using a modified Balzers 300 system. Examination in stereo of replicas of the microtubule‐containing marginal band revealed filaments projecting from the microtubule walls to form links between adjacent microtubules. These cross‐bridging proteins may bundle the microtubules into the configuration of the marginal band (MB) and may also serve to stabilize the structure. The MB appears to have linkages to components of the surface‐associated cytoskeleton (SAC). The SAC forms a continuous matrix that spreads across the upper and lower surfaces of the cell adjacent to the plasma membrane and extends around the outer perimeter of the MB. Thus, the SAC encapsulates the MB and the central nucleus. After lysis, the elements of the cytoskeleton remain in a configuration similar to that found in the whole cell. Spectrin (fodrin) and actin were identified by immunofluorescence in the region of the SAC. When labeled with antibodies specific for vimentin and synemin, a network of intermediate filaments can be detected in the region between the nucleus and the MB. These vimentin filaments are also enclosed within the SAC and appear in Pt‐C replicas to emerge from the area of the nuclear envelope. As the filaments extend toward the periphery of the cell, they form attachments to the SAC. Attachments of intermediate filaments to both the nucleus and the SAC thus appear to anchor the nucleus in its central position within the cytoskeleton.
Polarized microtubule gliding and particle saltations produced by soluble factors from sea urchin eggs and embryos
Authors: Pryer Nancy K. , Wadsworth Patricia , Salmon E. D.
Published: February 2005

In this report, we describe an in vitro system for analyzing microtubule‐based movements in supernatants of sea urchin egg and embryo homogenates. Using video enhanced DIC microscopy, we have observed bidirectional saltatory particle movements on native taxol‐stabilized microtubules assembled in low speed supernatants of Lytechinus egg homogenates, and gliding of these microtubules across a glass surface. A high speed supernatant of soluble proteins, depleted of organelles, microtubules, and their associated proteins supports the gliding of exogenous microtubules and translocation of polystyrene beads along these microtubules. The direction of microtubule gliding has been determined directly by observation of the gliding of flagellar axonemes in which the (+) and (–) ends could be distinguished by biased polar growth of microtubules off the ends. Microtubule gliding is toward the (−) end of the microtubule, is ATP sensitive, and inhibited only by high concentrations of vanadate. These characteristics suggest that the transport complex responsible for microtubule gliding in S2 is kinesin‐like. The implications of these molecular interactions for mitosis and other motile events are discussed.
Calmodulin and caimodulin‐binding proteins in the morphological transformation of sea urchin coelomocytes
Authors: Venuti Judith M. , Edds Kenneth T.
Published: February 2005

Sea urchin coelomocytes contain an actin‐based cytoskeleton that undergoes major organizational changes as the cells transform from one morphology (petaloid) to another (filopodial). The molecular mechanisms directing and regulating this cytoskeletal reorganization are not well understood; Ca2+ has been implicated, but the specific targets of its action have not been identified. Since the effect of Ca2+ on a variety of cellular processes has been shown to be mediated by the Ca2+‐binding protein calmodulin, we investigated the role of this protein in coelomocyte morphological transformation. The calmodulin inhibitory drugs trifluoperazine, chlorpromazine, and calmidazolium were shown to inhibit coelomocyte morphological transformation in response to hypotonic shock in a dosedependent fashion and at concentrations consistent with their reported potencies as anti‐calmodulin agents. Calmodulin isolated from coelomocytes using trifluorophenothiazine affinity chromatography co‐migrates with bovine brain calmodulin on 15% SDS‐polyacrylamide gels and demonstrates a characteristic shift in electrophoretic mobility in the presence of Ca2+. Another diagnostic for calmodulin, Ca2+‐dependent activation of exogenous 3':5' cyclic AMP phosphodiesterase, was demonstrated by whole coelomocyte homogenates, heat‐treated homogenates, and the affinity purified coelomocyte protein. Localization of calmodulin in coelomocytes by indirect immunofluorescence reveals an association of calmodulin, at least in part, with the actin‐based cytoskeleton. Calmodulin‐binding polypeptides with estimated relative mobilities of 240,000, 195,000, 170,000, 70,000, 60,000, 30,000, and 20,000 daltons were identified using 125I‐calmodulin overlay procedures. Ca2+‐dependent calmodulin‐binding in these preparations was demonstrated for all but the Mr 30,000 and 20,000 coelomocyte polypeptides. The majority of the calmodulin‐binding proteins identified in whole petaloid coelomocyte preparations are also found in Triton X‐100 insoluble cytoskeletal fractions. Immunoblotting with antiserum raised against chicken erythrocyte alpha‐spectrin suggests that the 240,000 Mr calmodulin‐binding polypeptide corresponds to coelomocyte alpha‐spectrin. This protein was enriched in isolated coelomocyte filopodia where, we propose, it serves an analogous function to its counterpart in erythrocytes, in linking the actin‐cytoskeleton to the plasma membrane. Thus, calmodulin is present in coelomocytes and possibly participates in the morphological transformation of these cells through regulation of cytoskeletal and/or membrane‐cytoskeletal interactions.
Antigenic interrelationship between the 40‐kilodalton cytokeratin polypeptide and desmoplakins
Authors: Gigi‐Leitner Orith , Geiger Benjamin
Published: February 2005

We describe here antigenic cross‐reactivity between the human 40‐kilodalton cytokeratin polypeptide [Moll et al] and components of bovine desmosomal plaque, namely desmoplakins I and II. This relationship was revealed by an antibody (KM 4.62), raised against cytoskeletal preparation of cultured human breast adenocarcinoma cells (MCF‐7) and selected by immunoblotting and immunofluorescent labeling. In cultured human cells that contain the 40‐kD cytokeratin, antibody KM 4.62 labeled arrays of filaments throughout the cytoplasm. This antibody labeled the basal layer of nonkeratinizing squamous epithelia as well as various simple (normal and malignant) epithelia and epithelial elements of the thymus. In liver tissue, labeling was obtained only in bile ducts and canaliculi but not in the hepatocytes.

In bovine cells and tissues, on the other hand, immunofluorescent labeling with antibody KM 4.62 was strictly confined to desmosomes. This was verified by double immunolabeling with both antibody KM 4.62 and specific cytokeratin or desmosomal antibodies. Immunoblotting analysis indicated that the former antibody reacts specifically with the high molecular weight components of the bovine desmosomal plaque, namely desmoplakins I and II. These immunochemical results suggest that bovine desmoplakins share same structural relationship with the human acidic, 40‐kD cytokeratin.
Evidence for regulation of lamellipodial and tail contraction of glycerinated chicken embryonic fibroblasts by myosin light chain kinase
Authors: Cande W. Zacheus , Ezzell Robert M.
Published: February 2005

Permeabilized cell models of muscle and nonmuscle cells have proven useful for examining the regulation of actin, myosin, and other cytoskeletal proteins during cell contraction. Upon addition of Ca2+ and ATP, glycerinated chick embryonic skin fibroblasts retract their tails and lamellipodia. Ca2+‐independent contractions are obtained by preincubation of cell models in Ca2+ ATPγS, followed by EGTA and ATP addition, or by addition of trypsin‐treated myosin light chain kinase that no longer requires Ca2+ for reactivation. By pretreating cells before glycerination with colchicine, it is possible to study lamellipodial contraction independent of tail contraction. Similar responses to ATPγS pretreatment and unregulated myosin light chain kinase are observed in cells that only contain lamellipodia. SDS‐PAGE electrophoresis of glycerinated fibroblasts incubated in ATPγ35S and Ca2+ shows that only two major proteins are thiophosphorylated, and that one of them, a band that comigrates with the 20K MW light chain of myosin, is thiophosphorylated in a Ca2+‐dependent manner. Since the rate of tail contraction is several‐fold faster after Ca2+ and ATPγS pretreatment or incubation in excess myosin light chain kinase, myosin light chain phosphorylation may be a rate‐limiting step during contraction.
Actomyosin dynamics in chemotactic amoeboid movement of Dictyostelium
Authors: Fukui Yoshio , Yumura Shigehiko
Published: February 2005

Under some physiological conditions, amoeboid cells are known to display a vectorial movement in response to chemotactic signals. Recent cell biological studies demonstrated the establishment of myosin filaments in Dictyostelium and indicate that the site of the motive force production is primarily localized at the posterior cortex. Furthermore, the traction force at the side body of a polarized amoeba is thought to play an essential role for the amoeboid movement. It is also suggested that certain dynamic activities of the actomyosin‐based cytoskeletal system are related to the establishment of mechanochemical transducing elements transiently organized during the chemotaxis. This review focuses on recent selective studies on the chemotactic movement of Dictyostelium, which is coupled with rapid redistribution of myosin filaments, and probably mediated by a transient phosphorylation of myosin.
Quinacrine‐induced changes in mitotic PtK1 spindle microtubule organization
Authors: Armstrong Lydia , Snyder Judith Armstrong
Published: February 2005

Quinacrine, an acridine derivative which competitively binds to ATP binding sites, has been used to study the role of ATP requiring molecules in microtubule organization in mitotic PtK1 cells. Brief treatments of metaphase cells with concentrations of quinacrine ranging from 2 to 10 μM decreased spindle length and birefringence in a concentration‐dependent manner. With either increasing quinacrine concentrations or duration of treatment, metaphase cells demonstrated a specific reorganization of spindle microtubules. Both polarization and electron microscopy showed a substantial loss of non‐kinetochore spindle microtubules with an increase in astral microtubules: this was particularly evident in the region adjacent to the spindle domain. Addition of millimolar concentrations of dinitrophenol to quinacrine‐containing medium did not potentiate the response of metaphase cells to quinacrine treatment. Time‐lapse video analysis demonstrated that the astral microtubules are the result of reorganization of spindle microtubules. These data suggest that functional ATP binding sites are required to maintain stable interactions between microtubules and that these interactions are responsible for maintaining the bowed configuration of non‐kinetochore spindle microtubules which are under compression at metaphase.
Spindle function in the green alga Mougeotia: Absence of anaphase A correlates with postmitotic nuclear migration
Authors: Pickett‐Heaps Jeremy D. , Wetherbee Richard
Published: February 2005

In the filamentous green alga Mougeotia, each daughter nucleus formed by mitosis is then rapidly moved along the recently divided daughter cell to the central cleavage developing in the chloroplast. This movement is brought about by a cone‐shaped array of microtubules (MTs) that ensheath the daughter nucleus and are focused upon a small region, presumably a microtubule‐organising center (MTOC). Movement is completed when the MTOC locates and then resides in the chloroplast cleavage, drawing the nucleus into this position.

The mitotic spindle is open with initially broad, ill‐defined poles. Anaphase A contributes minimally, if at all, to chromosome separation since the half spindles remain about the same length during anaphase and telophase. Thus, anaphase is accommodated and probably achieved by spindle elongation; the interzonal MTs also generate a rudimentary phragmoplast at the ingrowing cleavage furrow. The persistent polar MTs become directly transformed into the cone‐shaped array and initiate nuclear movement during early telophase. Various closely or distantly related green algae show this trait of persistent polar MTs. We conclude that this trait has allowed some species to evolve a motility system based directly on the capabilities of astral MTs, for generating the postmitotic nuclear movement essential for the restoration of the interphase cell organization.
Massive actin bundle couples macrocilia to muscles in the ctenophore Beroë
Authors: Tarmm Sidney L. , Tamm Signhild
Published: February 2005

Macrocilia are thick compound ciliary organlles arising individually from elongated epithelial cells on the lips of beroid ctenophores. A giant wedge‐shaped bundle of microfilaments extends 25–30 μm from the base of each macrocilium to the lower end of the cell, terminating at a junction with an underlying smooth muscle cell. The broad end of the microfilament bundle is anchored to the macrocilium by striated rootlet fibers that extend from the basal bodies into the bundle and are linked to the microfilaments by periodic bridges. Fluorescence microscopy of rhodamine‐phalloidin stained intact tissue, dissociated macrociliary cells, and Triton/glycerol‐isolated bundles shows that the microfilaments contain actin. The microfilaments run generally parallel to the long axis of the bundle but are not highly ordered. Filaments decorated with myosin S1 show a uniform polarity with arrowheads pointing away from the tapered membrane‐associated end of the bundle. No variations in bundle length (nor changes in rootlet periodicity) were observed in tissue fixed under conditions of calcium activation. Isolated bundles did not contract in Mg‐ATP, even though detached macrocilia underwent reactivated beating and sliding disintegration. Macrocilia arc used to bite through food organisms or transport prey into the stomach. The actin filament bundles probably play a supporting role as a structural linker between macrocilia and subepithelial muscle fibers and may serve as intracellular tendons lo mechanically coordinate the motor activities of macrocilia and muscles during prey ingestion.
Translational control and the cytoskeleton in Physarum polycephalum
Authors: Brodeur Richard D. , Jeffery William R.
Published: February 2005

Translationally active plasmodia of the syncytial slime mold Physarum polycephalum develop into translationally dormant sclerotia during starvation. Although functional mRNA and ribosomes exist in sclerotia, protein synthesis is suppressed at the level of initiation. To test the possibility that alterations in the cytoskeleton may limit protein synthesis, we have examined the distribution of polysomes and actin mRNA in the cytoskeletal (CSK) and soluble (SOL) fractions of Triton X‐100‐extracted plasmodia and sclerotia. Most of the polysomes and actin mRNA were located in the CSK of plasmodia, while most of the ribosomes and actin mRNA were located in the SOL of sclerotia. The results suggest that ribosomes and mRNA shift from the CSK to the SOL as protein synthesis is suppressed during starvation. Plasmodia and sclerotia can be induced to accumulate excess polysomes by treatment with low levels of the elongation inhibitor cycloheximide. Treatment of plasmodia with cycloheximide caused excess polysomes to accumulate in the SOL, suggesting that the CSK contains a limited capacity for binding translational components and that the association of polysomes with the cytoskeleton is not required for protein synthesis. Treatment of sclerotia with cycloheximide, however, caused polysomes and actin mRNA to accumulate in the CSK, suggesting that the selcrotial cytoskeleton, although depleted in ribosomes and mRNA, is capable of binding translational components. It is concluded that alterations in the sclerotial cytoskeleton are not involved in translational control.
High time‐resolution analysis of transient bending patterns during ciliary responses following electric stimulation in sea urchin embryos
Authors: Baba Shoji A. , Mogami Yoshihiro
Published: February 2005

Transient ciliary movement during responses to electric stimulation of embryos of the sea urchin, Hemicentrotus pulcherrimus, was analyzed in terms of angular direction with a time resolution of approximately 2 ms with high‐speed microcinematography. In the primitive response, which can be induced only in the early stages of development of the embryo, bending transients always started with a short pause in the middle of the effective stroke, irrespective of beat position on stimulation. In the reversal response, induced only in the late stages of development, bending transients occurred with a delay as short as some 10 ms from stimulation, and with a transient sharp deviation from the normal beat before the cilium took the position of the beginning of the recovery stroke of the reversed beat. The delay was significantly shorter at the base than at the tip, suggesting that some form of signal travels along the cilium; the speed was ten times higher than that of propagating bends in the normal beat. These facts indicate that the sensitivity to internal changes resulting from stimulation of the axoneme may vary with development, ciliary beat positions, and regions along the cilium.
Movements of intracellular particles in undifferentiated amebae of Dictyostelium discoideum
Authors: Roos U.‐P. , De Brabander M. , Nuydens R.
Published: February 2005

We recorded live, undifferentiated amebae of Dictynstelium discoideum by video microscopy and analyzed the behavior of cytoplasmic particles and granules. Cytoplasmic streaming and saltatory movements are the two major types of particle movements that occur in interphase amebae. Saltatory movements predominated in an area around the nucleus‐associated body (NAB) and many were radial toward or away from it, the velocity being very similar in both directions. Some saltations were simple forward movements, and others were complex to‐and‐fro movements with as many as seven turnabouts. For a given leg of movement the velocity was not uniform along the path. Small particles (< 1 μm) moved faster (X = 2.8 μm/s) than large (∼ 1 μm; X = 2.1 μm/s) and very large (> 1 μm; X = 1.4 μm/s) particles, but the smallest particles were visible only in the running image and could not be analyzed. Ultrastructurally, saltating particles are digestive vacuoles and vesicles of various sizes, appearances, and contents, which are numerous particularly in the vicinity of the NAB. Several lines of evidence pointed to a role of microtubules (MTs) in saltatory particle movements. Composites of particle tracks corresponded closely to MT arrays visualized by immunofluorescence. No saltations occurred in mitotic amebae that lack cytoplasmic MTs, but the movements resumed toward the end of division, concurreduced with the rebuilding of the complex of cytoplasmic MTs. Nocodazole reduced and eventually slopped saltatory movements over a period of 3 h, when aberrant MT patterns were the rule. Saltations in slime mold amebae may be an eye‐catching feature of intracellular transport functioning in endo‐ and exocytosis in the shuffling of vesicles containing factors involved in ameboid movement, and in the transduction of external signals to the cell center.
Isolation and characterization of sea urchin egg spectrin: Calcium modulation of the spectrin–actin interaction
Authors: Fishkind Douglas J. , Bonder Edward M. , Begg David A.
Published: February 2005

Sea urchin egg spectrin has been purified from a homogenate of unfertilized Strongylocentrotus purpuratus eggs using standard biochemical procedures. SDS‐PAGE analysis of the molecule revealed a closely spaced, high molecular weight doublet at 237/234 kDa (present in an equimolar ratio). Rotary shadowed images of egg spectrin revealed a double‐stranded, elongate, flexible rod‐shaped contour, measuring 210 nm in length and ∼ 4–8 nm in width. Additionally, this molecule is shown to be immunologically related to avian erythroid spectrin, since it cross‐reacts with antibodies prepared against the chicken erythrocyte α‐spectrin/240 kDa subunit. The interaction of egg spectrin with actin was examined by sedimentation and falling‐ball viscometry assays. The binding and cross linking properties of spectrin to actin demonstrate a unique Ca++‐sensitive regulation at micromolar Ca++ concentrations. This observation provides new insight into the way Ca++ may regulate spectrin–actin interactions in vitro and further suggests possible structural and modulatory roles for egg spectrin in the developing sea urchin embryo.
Organization and dynamics of a cortical fibrous network of Paramecium: The outer lattice
Authors: Cohen Jean , de Loubresse Nicole Garreau , Klotz Catherine , Ruiz Françloise , Bordes Nicole , Sandoz Daniel , Bornens Michel , Beisson Janine
Published: February 2005

A monoclonal antibody, CC212, raised against ciliated cortices of quail oviduct cells and characterized as an antimyosin of smooth muscle and nonmuscle cells, was shown to specifically label a regular cortical network in Paramecium and to recognize two Triton X‐100–insoluble polypeptides at 130 and 50 kDa. However, no evidence was obtained that these polypeptides are related to myosin.

An immunofluorescence study and ultrastructural immunogold localization allowed us to identify the CC212‐decorated material as a component of the outer lattice, a submembrane cytoskeletal network which runs along the top of the ridges visible by scanning electron microscopy and delineates the periphery of each cortical unit. The dynamics of the outer lattice during the cell cycle was studied by immunofluorescence and it was found that the outer lattice growth is achieved without disruption of the preexisting meshes by longitudinal elongation and additon of new transverse partitions. A striking disorganization of the outer lattice was observed in a thermosensitive mutant primarily altered in basal body duplication. These observations suggest possible functions of the outer lattice and demonstrate the interdependence of basal body duplication, surface growth, and outer lattice remodelling.