bluuebunny/crossref_metadata_2025 huggingface dataset:
Coupling of photomovement and photosynthesis in desmids
Authors: Häder Donat‐P.
Published: February 2005

The effects of the uncouplers CCCP and DNP on photokinesis, phototaxis, and the photophobic response in the desmid Cosmarium have been studied both in population systems and by videomicrographic, single‐cell analysis. Light‐dependent motility is specifically inhibited by both uncouplers, indicating that photokinesis is driven by photophosphorylation. In population experiments, phototaxis and accumulations in light traps due to photophobic responses are inhibited by drug concentrations comparable to those that inhibit photokinesis. Analysis of single‐cell behavior demonstrated, however, that neither photophobic responses elicited by an increase in light intensity (step‐up response) nor by a decrease (stepdown response) are inhibited, as long as the reduced motility allows the organisms to cross a light‐‐dark border. Phototactic orientation is not impaired by DNP in the single cell analysis, but CCCP significantly reduced the degree of orientation. The results indicate that, although chlorophyll is the photoreceptor for all three photoresponses, at least the photophobic response is independent of both the photosynthetic electron transport chain and photophosphorylation.
Effects of phalloidin microinjection and localization of fluorescein‐labeled phalloidin in living sand dollar eggs
Authors: Hamaguchi Yukihisa , Mabuchi Issei
Published: February 2005

Effects of microinjection of phalloidin on fertilization and cleavage of sand dollar (Clypeaster japonicus and Scaphechinus mirabilis) eggs were studied. The drug, previously injected into unfertilized eggs, showed no effect on the elevation of the fertilization membrane upon insemination up to an intracellular concentration of 50 μM. However, the movement of the egg pronucleus to the sperm pronucleus was inhibited and the fusion of pronuclei did not occur. The subsequent development no longer took place. When phalloidin was injected into fertilized eggs, the thickness of the cortical layer increased and the microvilli became conspicuous. Both nuclear division and cleavage were inhibited at the intracellular concentration of more than 20 μM, though the latter seemed to be more sensitive to phalloidin than the former.

Fluorescein‐labeled phalloidin (FL‐phalloidin) was injected into eggs in order to investigate F‐actin localization by fluorescence microscopy. In both unfertilized and fertilized eggs, FL‐phalloidin was localized in the cortical layer within 1 min after injection. It was also localized in the cortical layer as radially oriented rodlike structures when injected into fertilized eggs before the disappearance of the nuclear membrane. No distinct fluorescence was detected in the mitotic apparatus or in the cleavage furrow. FL‐phalloidin redistributed gradually into egg cytoplasm. In unfertilized eggs, fluorescent rods were found especially in the egg pronucleus 30 min after injection.
Organization of the cytoskeleton in square fibroblasts
Authors: Marek L. F. , Kelley R. O. , Perdue B. D.
Published: February 2005

The relationship between the cytoskeleton, stress fiber formation, and cell shape has been difficult to determine in fibroblasts grown in tissue culture. Vagaries in cell shape are complicated, as well, by stochastic cell movements. We dictated the attachment sites and shape of fibroblasts by growing them on square adhesive substrates surrounded by nonadhesive substrates. Cytoskeletal models were made by treating the cells with buffered Triton X‐100 and glycerol. The residues were then examined by scanning electron microscopy followed by light microscopy of the same cells. The cytoskeletons of randomly moving cells were examined with whole mount transmission microscopy to confirm images seen with scanning microscopy. The cells thus examined demonstrated definite relationships between ruffling activity and stress fiber terminations, which were limited to the more adhesive, palladium substrate. No stress fibers were seen to end on the lesser adhesive substrate, agarose, and ruffling did not occur across the agarose. Cells too small to fill an entire square tended to extend across one diagonal of the square, and the stress fibers ran parallel to the longest axis of these cells. Larger cells were able to completely fill their squares. The cytoskeletons of these cells were organized in a spatial relation to the square shape of the cells. The cortical meshwork was aligned circularly and diagonally within the cells. Stress fibers appeared to form from the microfilaments of the meshwork and were aligned diagonally across the cells. We conclude that the diagonal arrangement of the stress fibers and cortical meshwork is caused by the same mechanism by which smaller cells spread over the longest axis of a square. Regions of cells where the meshwork was absent or where stress fibers were tightly bundled were occupied by more randomly arranged cytoskeletal components. Regions of tighyly bundled stress fibers did not seem to coincide with regions of cortical meshwork as seen by either whole mount transmission or scanning electron microscopy. Stress fibers were revealed in the light microscope to course beneath more randomly oriented cytoskeletal elements. These “lacework‐like” elements were found frequently in square cells. Conspicuous structures in this random lacework were focal points of radially arranged filaments. Our observations suggest a continuity between stress fibers and the cortical microfilaments. The orientation of fibers and filaments was, in turn, dependent on cell shape for organization within the cell.
Cell locomotion in vitro by Xenopus laevis gastrula mesodermal cells
Authors: Nakatsuji Norio , Johnson Kurt E.
Published: February 2005

Prospective mesodermal cells of Xenopus laevis gastrulae showed substantial locomotion in vitro, averaging 4.3 μM/min, when dissociated and cultured on a glass surface coated with collagen and fetal bovine serum. The cell translocate by making lamellipodia and filopodia whereas the main cell body remains rounded. When two mesodermal cells made contact with each other, they showed contact paralysis of lamellipodial activity. In contrast, when mesodermal cells contact ectodermal cells, contact paralysis does not occur. Rather, migrating mesodermal cells continue to translocate. The locomotion in vitro appears to mimic that in vivo during gastrulation, because of the similarities of the rate of movement and the cell shape in culture and in embryos. Neither prospective ectodermal cells from gastrulae nor prospective mesodermal cells from blastulae showed locomotion under the same culture conditions.
Capping of cross‐linked concanavalin a receptors on the surface of substrate‐spread platelets
Authors: Alexandrova A. Y. , Vasiliev J. M.
Published: February 2005

Ligand‐induced redistribution of concanavalin A (Con A) receptors on the surfaces of substrate‐spread rabbit platelets was studied. These receptors were diffusely distributed on the surface of prefixed cells. Incubation of living cells with Con A and anti‐Con A antiserum led to redistribution of corresponding receptors: These receptors were patched, removed from the upper surface of substrate‐attached pseudopodia and lamellipodia, and accumulated on the surface of central cell parts. Capping of receptors was inhibited by cytochalasins B and D and by cold. Thus, capping of surface receptors can take place not only in large nucleated cells but also in platelets, that is, in small anucleate cell fragments. It is suggested that the cells of various types in the course of their attachment to the substratum form pseudopodia and lamellipodia that have a number of common characteristics, including the ability to move the cross‐linked surface receptors centripetally.
Directional protrusive pseudopodial activity and motility in macrophages induced by extracellular electric fields
Authors: Orida Norman , Feldman Joseph D.
Published: February 2005

Extracellularly applied electric fields (< 12 V/cm) strongly influence murine resident peritoneal macrophages (Mø) to undergo directional protrusive pseudopodial activity to wards the positive pole of the electric fields in the absence of exogenously applied chemotactic ligands. Internal and external morphological features were not grossly disrupted by the fields. Directional motility induced by the electric fields was inhibited in the presence of 1.0 mM La3+ or 2.5 mM Mg2+ and 5.0 mM EGTA. Effects of the fields were latent in the inhibited cells and directional motility was expressed after termination of the field and removal of the inhibitors. Receptors for the lectins concanavalin A (Con A) and phytohemagglutinin (PHA‐L) were uniformly distributed on the surfaces of Mø with no exposure to electric fields. After exposure to the fields, Con A receptors were preferentially distributed on regions of the Mø surface facing the negative pole and PHA‐L receptors were preferentially distributed on those regions facing the positive pole. The possibility that directional Mø motility is regulated by the molecular topography of the cell surface is discussed.
Persistence of the expression of β‐tropomyosin in dystrophic avian pectoral muscle
Authors: Feit Howard , Domke Robert
Published: February 2005

The isotype pattern of tropomyosin was investigated in normal and dystrophic avian pectroal muscle using two‐dimensional gel electrophoresis. Previous reports have shown that adult pectoral muscle of chickens contains only the α‐subunit of tropomyosin and a breast‐type troponin‐T (TN‐T), whereas pectoral fetal muscle contains both α‐ and β‐tropomyosin and leg‐type TN‐T. The change from the fetal to the adult forms begins shortly after hatching. It has been previously reported that avian dystrophic pectoral muscle contains both the leg‐ and breast‐type TN‐T; we show that in avian dystrophic muscle there is also persistent expression of the β‐subunit of tropomyosin.
Effect of various extraction solutions and thrombin activation on the composition of the platelet cytoskeleton
Authors: Rosenberg Sharon , Lawrence John , Stracher Alfred
Published: February 2005

When human blood platelets were immersed in an ice‐cold solution containing 1% Triton ×‐1200, 40 mM KCl, 10 mM EGTA, 10 mM imidazole‐HCl, and 2 mM NaN3 pH 7.0, a flocculent precipitate appeared immediately in the tube. This precipitate was collected at 3,000g and SDS‐polyacrylamide gel analysis showed it to consist mainly of actin, α‐actinin, actin‐binding protein (ABP), and varying amounts of myosin.

Any modifications of this solution used to isolate the platelets' Triton‐insoluble cytoskeleton caused profound changes in the nature of the cytoskeleton isolated. Increasing the KCl concentration resulted in a lower yield of cytoskeletal actin and ABP. Inclusion of EDTA in the solution resulted in an increased amount of myosin associated with the cytoskeleton, whereas including MgATP decreased the myosin yield.

Experiments with the purified proteins showed that ABP and myosin can each protect the actin from depolymerizing when dialyzed into the Triton solubilization solution. In addition, it was found that when platelets were stimulated with thrombin for 2 min prior to the addition of the Triton solution, 3–4 times more myosin was associated with the cytoskeletal precipitate.

The results suggest, therefore, that any variations in solution conditions used for isolating the cytoskeleton from resting platelets, which results in alterations in the amount of ABP, may have profound effects on the state of actin polymerization. Likewise, in thrombin‐activated platelets, it is suggested that the increased association of myosin with the cytoskeleton results in a greater stabilization of the F‐actin associated with the cytoskeleton. These factors must be considered when interpreting the results regarding the nature of actin transformations in the resting and activated platelet.
Effect of fragmin on actin polymerization: Evidence for enhancement of nucleation and capping of the barbed end
Authors: Sugino Hiroaki , Hatano Sadashi
Published: February 2005

As reported previously, fragmin isolated form Physarum plasmodia restricts the polymerization of actin to produce short F‐actin filaments in the presence of Ca2+ ions. Here it is shown that when actin is polymerized at low concentrations of salts, fragmin increases the critical concentration of actin for polymerization. This effect of fragmin on the critical concentration is independent of the molar ratio of fragmin to actin. The addition of actin monomers onto heavy meromyosin‐decorated F‐actin fragments treated with fragmin occurs unidirectionally at the pointed end of each fragment. These results suggest that fragmin binds to the barbed ends of F‐actin filaments and inhibits association and dissociation of actin monomers at this end. Fragmin accelerates the initial stage of polymerization of actin. When a constant amount of G‐actin is polymerized in the presence of small amounts of fragmin, the inverse of the half‐polymerization time increases in proportion to the square root of the amount of fragmin added. This means that fragmin acts as a potent promoter of the nucleation step in actin polymerization. Both functions of fragmin–promotion of nucleation and capping at the barbed end of F‐actin–require micromolar concentrations of Ca2+.
Evidence for a role of 13S axonemal ATPase in modulation of ciliary microtubule sliding
Authors: Zanetti Nina C. , Warner F. D.
Published: February 2005

We recently demonstrated that elevated concentrations (≥ 20 μM) of the dynein substrate MgATP2− inhibit the spontaneous ATP‐induced sliding disintegration of isolated, Triton‐demembranted Tetrahymena cilia. We have used a turbidimetric assay (ΔA350 nm) and electron microscopy to examine the effect of ATP on sliding disintegration when activated by other divalent cations. Mg2+, Ca2+, and Mn2+ are each capable of activating sliding, but only with Mg2+ and Mn2+ is disintegration inhibited by elevated ATP concentrations (≥ 1 mM). The two major ATPase activities obtained by KCI extraction of Tetrahymena axonemes differ in their cation specificities such that Mg2+ and Ca2+ activate the 21S dynein ATPase with equal efficiency, whereas the 13S axonemal ATPase activity is reduced by ∼ 50% when CaATP2− replaces MgATP2− as substrate. With 1 mM MgATP2− as substrate, 10−7 to 10−2M added CaC12 alleviates the ATP‐dependent inhibition of disintegration and likewise represses 13S MgATPase activity. In contrast, free Ca2+ has no effect on either the disintegration response or MgATPase activity. In contrast to Triton‐treated cilia, glycerinated cilia, which beat in 1 mM MgATP2−, are inhibited from beating by high CaATP2− concentrations. These substrate specificities suggest that concentration‐dependent, substrate inhibition of sliding disintegration may be a manifestation of a physiological mechanism that is mediated by the 13S axonemal ATPase and that may function to modulate sliding during bend formation. However, the effects of added CaCl2 probably do not reflect a physiological mechanism for regulating beat parameters, but rather may result from CaATP2− competing for MgATP2− binding sites on the 13S ATPase, thereby blocking expression of the 13S ATPase.
Taxol binds differentially to flagellar outer doublets and their reassembled microtubules
Authors: Parness Jerome , Asnes Clara F. , Horwitz Susan Band
Published: February 2005

Taxol induces the in vitro assembly of calcium stable microtubules from flagellar tubulin solubilized from sea urchin (Strongylocentrotus purpuratus) sperm tail outer doublets by sonication. Assembly occurs in the presence or absence of exogenous GTP. The drug (10 μM) reduces the critical concentration of protein required for assembly to ⩽0.04 mg/ml. 3H‐Taxol binds specifically to both isolated flagellar outer doublets and to reassembled microtubules with calculated maximal binding ratios of 0.25 and 1.32 moles taxol/mole polymerized flagellar tubulin dimer, respectively. We suggest that the discrepancy in maximal binding ratios may result from the presence of an endogenous molecule(s) along the surface of outer doublet microtubules that restricts taxol binding to that structure.
Characterization of the chlamydomonas flagellar collar
Authors: Snell William J.
Published: February 2005

The flagella of Chlamydomonas reinhardtii protrude through the cell wall via short, tunnel‐like openings that are lined with 11 nm × 500 nm fibers arranged in parallel array. These cylindrical collections of fibers presumably permit free movement of the flagella within the cell wall. In this report electron‐microscopic evidence is presented showing that during the initial stages of the mating reaction intact collars slip off of the ends of the flagella when cell wall loss occurs. Electrophoretic analysis of isolated collars reveals one major protein and several minor species.
Crawling caenorhabditis elegans spermatozoa contact the substrate only by their pseudopods and contain 2‐nm filaments
Authors: Roberts Thomas M.
Published: February 2005

The locomotion of C. elegans spermatozoa resembles, in many respects, the crawling movements of other eukaryotic cells. However, these sperm contain surprising little actin, which plays no apparent role in this cell's motility. Electron microscopy has revealed that crawling spermatozoa retain a strict morphological polarity so that the organelle‐filled cell body is separated from the pseudopod by an array of cytoplasmic laminar membranes. When sperm crawl only the pseudopod contacts the substrate; the cell body is either pulled behind or carried on top of the rear portion of the pseudopod. Fingerlike projections which extend forward from the leading edge of the pseudopod initiate contact with the substrate. The underside of the pseudopod exhibits areas of close (40 nm separation) membrane‐substrate association with intervening areas of wide (up to 300 nm) membrane‐substrate gaps. The pseudopod cytoplasm contains 2‐nm filaments but no filamentous actin has been observed. These 2‐nm filaments were detected in thin sections of crawling cells and in negative‐stained remnants of spermatozoa disrupted by either hypotonic buffer on Triton X‐100. The filaments are found both free in the cytoplasm and closely associated with the cytoplasmic face of the plasma membrane and are usually oriented along the long axis of the cell. Neither the identity nor the function of these filaments has been established although their location and orientation suggest that they may be involved in generating propulsion.
Microfilament rearrangements during fibroblast‐induced contraction of three‐dimensional hydrated collagen gels
Authors: Farsi Jamila M. A. , Aubin Jane E.
Published: February 2005

In vitro models have been developed recently to study the ability of fibroblasts to generate tensile force within collagen gels. The present study was initiated to assess the role of the cytoskeleton in the cell shape changes and force generation in one such model system. Porcine periodontal ligament fibroblasts (PPLF) were cultured within three‐dimensional collagen gels attached to glass coverslips. Fluorescence microscopy, using nitrobenzooxadizole (NBD)‐phallacidin labeling for microfilaments and tubulin antibody staining for microtubules, was combined with phase and Nomarski optics to determine the intra‐ and extracellular architecture of the cells and collagen fibers. Samples were observed from 30 minutes to 24 hours after initiation of cell attachment. During attachment and spreading, NBD‐phallacidin staining changed dramatically until large microfilament bundles became prominent. Collagen fiber alignment, compaction, and finally tearing from the coverslip occurred during this time. After release of tension, microfilament bundles were no longer evident. The change in microtubule distribution during these processes was less dramatic, appearing to follow the change in cell shape. These results indicate that microfilaments play an essential role in generating force to align and compact collagen, while microtubules may have a secondary role only.
An unusual mechanism of cell contraction: Leptodiscinae Dinoflagellates
Authors: Cachon Jean , Cachon Monique
Published: February 2005

The Leptodiscinae, a group of marine Dinoflagellates, are good material for the study of contraction though they cannot be collected in abundance. Their cell bodies are flattened anteroposteriorly (Leptodiscus, Leptophyllus, and Leptospathium) and are able to contract suddenly when the surrounding water is disturbed.

Electron microscopical observations have shown that the structures responsible for the contraction consist of a layer of parallel filaments located beneath the cell membrane of some specialized parts of the body. These filaments seem to be nonactin (NAF) because of their diameter (2.5–3 nm) and because they are not decorated by heavy meromyosin (HMM). They appear helically coiled and doubly twisted, and form tubular structures when contracted.
Dynamics of keratin filaments and the intermediate filament distribution center during shape change in PtK1 cells
Authors: Eckert Barry S. , Caputi Susan E. , Warren Robert H.
Published: February 2005

Reorganization of intermediate filaments during cell spreading is examined by immunofluorescence, electron microscopy, and time‐lapse video microscopy. A juxtanuclear cap, believed to correspond to the intermediate filament distribution center, was observed to be spatially related to the organization of the intermediate filament network as cells spread. A keratin cap was observed, which appeared spontaneously in motile PtK1 cells. Cap formation may be a consequence of retraction of intermediate filaments from the cytoplasm as cells move. The position of this juxtanuclear cap is related to the direction of movement, located on the side of the nucleus near the advancing edge of the cell. As the cell spreads, the cap disappears as the keratin filament network returns to the cytoplasm. Evidence presented here is consistent with the hypothesis that the distribution center mediates keratin filament organization during cell shape change.
Evidence against surf‐riding as a general mechanism for surface motility
Authors: Bowser Samuel S. , Bloodgood Robert A.
Published: February 2005

The mechanism responsible for the energy‐dependent movement of membrane components (ie, surface motility) is unknown. Recently a potentially unifying model, termed “surf‐riding” [Hewitt, 1979] or “surf‐boarding” [Berlin and Oliver, 1982], has been proposed to explain surface motility. Using phase‐contrast light microscopy and membrane surface markers (polystyrene microspheres), we have tested the surf‐riding/surf‐boarding hypothesis on two protozoan systems: the axopodia of the heliozoan Echinosphaerium nucleofilum and the reticulopodial networks of the allogromiid foraminiferans Allogromia laticollaris and Allogromia sp, strain NF. Our evidence indicates that surface motility, as displayed by these organisms, does not occur by a surf‐riding/surf‐boarding mechanism. Previouś observations on surface motility associated with the Chlamydomonas flagellum indicate that this system is also incompatible with the surf‐boarding/surf‐riding hypothesis.
Cytoplasmic tubulin from the unfertilized sea urchin egg: II. Variation of the intrinsic calcium sensitivity of strongylocentrotus purpuratus egg tubulin as a function of temperature and brain microtubule‐associated proteins
Authors: Suprenant Kathy A. , Rebhun Lionel I.
Published: February 2005

Cytoplasmic tubulin purified from unfertilized sea urchin eggs self‐assembles in the absence of microtubule‐associated proteins (MAPs) [Suprenant and Rebhun, 1983; Detrich and Wilson, 1983] with a critical concentration for polymerization of 0.8 mg/ml at 15–18°C, a value well below the 3 mg/ml tubulin present in these eggs [Pfeffer et al, 1976]. Studies of the calcium sensitivity of unfertilized S. purpuratus (sea urchin) egg tubulin were initiated to help understand how this tubulin is maintained unassembled in the unfertilized egg. Egg microtubules, assembled at physiological temperatures (15–18°C) were depolymerized by a 100‐fold lower free calcium concentration than egg microtubules assembled at the higher temperatures (25–37°C) generally used to assemble mammalian brain microtubules. The initial rate of egg microtubule assembly was much more sensitive to calcium than was microtubule depolymerization at steady state at 37°C. However, both processes were sensitive to near physiological free calcium of free calcium for depolymerization than microtubules assembled at 18°C from egg tubulin alone. While calcium regulatory MAPs have not yet been found in sea urchin eggs, the fact that brain MAPs interact with egg tubulin and regulate both its critical concentration for polymerization [Suprenant and Rebhun, 1983] and its calcium sensitivty, suggests that such regulatory molecules exist. These results suggest that sea urchin egg tubulin assembly in vivo could be controlled by variations in interacellular calcium levels acting in concert with urchin egg proteins similar in function to brain MAPs.
Energy requirements for pigment aggregation in Fundulus melanophores
Authors: Clark Theodore G. , Rosenbaum Joel L.
Published: February 2005

Teleost chromatophores are filled with individual pigment granules that rapidly aggregate to the cell center or become dispersed throughout the cytoplasm in response to environmental stimuli. Microtubules appear to be required for pigment aggregation (movement toward the cell center), and recent findings have suggested that a dynein‐like ATPase may participate in force production. Based on previous studies, however, it has been argued that pigment aggregation does not require energy directly, a view that supports the involvement of an elastic component in granule movement. To examine this point further, we have reinvestigated the energy requirements for pigment aggregation using both intact cells and detergent‐permeabilized cell models of Fundulus melanophores. Poisons of oxidative phosphorylation, namely, 2,4 dinitrophenol and NaCN, reversibly inhibit melanosome aggregation in response to adrenaline. Inhibition of movement results directly from depletion of intracellular ATP, since pigment translocation can be reactivated in permeabilized cells by the addition of exogenous ATP to the lysis buffer. Non‐hydrolyzable analogues, including β,γ‐imidoadenosine‐5′‐triphosphate (AMPPNP), β,γ‐methylene adenosine‐5′‐triphosphate (AMPPCP), and ATPγS, will not substitute for ATP in reactivation of movement. Similarly, other nucleotides such as ADP, AMP, GTP, CTP, and ITP, have limited ability to support melanosome aggregation in metabolically poisoned cells subjected to detergent lysis. ATP itself has no effect on intact cells. These results indicate that melanosome aggregation is ATP‐dependent and energy‐driven, and are consistent with a role for a force‐transducing ATPase in particle movement.
Non‐actin filaments and cell contraction in Kofoidinum and other Dinoflagellates
Authors: Cachon Jean , Cachon Monique
Published: February 2005

The motility and fine structure of the marine planktonic dinoflagellate Kofoidinium and members of other related genera have been investigated. Several types of shape change were found to occur: slow morphogenetic changes (which also occurred as restitution movements in response to injury), movements associated with the ingestion of food and the evacuation of wastes, and more rapid movements concerned with the capture of prey. All these movements seemed to be brought about by the contraction of refractile tracts within the cytoplasm, which were found to contain 2.3‐nm filaments, some with a complex striated appearance. This and other evidence suggests that these filaments, which have counterparts in many other protists, are not actin filaments.
Study of the properties of MgATP2−‐induced stationary bends in demembranated sea urchin sperm
Authors: Sale Winfield S.
Published: February 2005

Methods of demembranation and reactivation of Lytechinus pictus sperm were developed that result in non‐motile sperm which take on a stable bend of about 3.5 radians at the proximal end of the cell. The middle and distal portion of the flagellum is relatively straight or slightly bent in the same direction forming a somewhat “C” shaped sperm cell. In these studies, we refer to this characteristic shape as the quiescent form, and as opposed to “rigor wave” sperm, the quiescent form is induced and maintained by a relatively high concentration of MgATP2− (> 0.2 mM). Other conditions important to the production and maintenance of the quiescent form in demembranated sperm include: starting with concentrated, undiluted sperm, maintaining low Ca++ in the demembranation buffer, using a minimum of 0.2 mM MgATP2− and pH of 7.9–8.1 in the reactivation buffer. Deviation from some of these conditions results in a dramatic increase in motile, asymmetrically beating sperm. Addition of 0.4 mM CaCl2 to the reactivation buffer increased the proximal bend angle to 5 radians. The induction and maintenance of the stationary bend is mediated by dynein activity: “rigor wave” sperm were transformed to the quiescent form upon 0.2 mM ATP addition; micromolar vanadate abolished the quiescent form by “relaxation” of the proximal bend; and the vanadate relaxed sperm were restored to quiescent form by catechol. Importantly, 20 μM cAMP activated motility of the otherwise quiescent‐form sperm. Quiescent‐form, demembranated sperm were also activated by mild trypsin digestion. These and other data suggest that the quiescent‐form sperm are trapped at the end of the principal bend, and these data are consistent with the proposal that the single stationary bend results from asymmetry of active microtubule sliding [Gibbons and Gibbons, (1980): J. Cell Biol. 84:13–27].
Tension in the culture dish: Microfilament organization and migratory behavior of quail neural crest cells
Authors: Tucker R. P. , Edwards B. F. , Erickson C. A.
Published: February 2005

We have investigated one aspect of the migratory behavior of quail neural crest (NC) cells by comparing the organization of microfilament bundles and the ability to distort migratory substrata by NC, somite, and notochord cells in vitro. In contrast to the numerous cytoplasmic stress fibers in somite‐derived fibroblasts and notochord cells revealed by rhodamine‐phalloidin staining and thin‐section electron microscopy, microfilaments in NC cells are restricted to the cell cortex. To test the relative degrees of tension generated by these cell types on the underlying substratum, cells were cultured in collagen gels and on distortable silicone rubber sheets. Explanted somites and notochords produced dramatic radial alignment of 750 μg/ml collagen gels, whereas neural crest cells only aligned gels of lower concentrations. Fibroblasts did not migrate individually from explanted somites and notochords into 250 μg/ml collagen gels as readily as into higher concentration collagen lattices. In contrast, neural crest cells migrated into matrices of low concentration as well as into higher concentration collagen gels. Neural crest cells and their pigmented derivatives did not distort silicone rubber sheets, whereas somite and notochord‐derived fibroblasts wrinkle this substratum after 4 days in culture. Thus, the differences in organization of the actin cytoskeleton reflect the tractional force exerted by these cells on their substratum. We hypothesize that the migratory behavior of NC cells in vivo may be related to their ability to translocate through embryonic extracellular matrices while generating relatively weak adhesions with the substratum, whereas the stronger forces generated by other embryonic cell types upon the delicate extracellular matrix may restrict their migration and may be associated with other morphogenetic events.
Isolation of newt lung ciliated cell models: Characterization of motility and coordination thresholds
Authors: Weaver Alice , Hard Robert
Published: February 2005

Demembranated ciliated cell models are useful for studying mechanisms responsible for the regulation of ciliary coordination and waveform. This paper describes procedures for isolating ciliated cells from the newt, Taricha granulosa, by trypsin dissociation, their subsequent demembranation by Triton X‐100, and their reactivation with MgATP to produce highly motile, coordinated, ciliated cell models. Reactivation of cell models with a high degree of mechanochemical coupling depended on avoiding mechanical damage and maintaining optimal conditions during all stages of isolation and reactivation. Highly motile models were prepared from cells incubated in trypsin, treated briefly with EDTA, separated by gentle agitation, and concentrated by centrifugation at low gravitational forces. Optimal demembranation and reactivation conditions were similar to those described previously for isolated newt lung axonemes. Under these conditions, nearly 100% of the models were reactivated when provided with MgATP and 90–95% beat with coordinated waves. The ciliary tufts beat at frequencies within the range measured in living cells and their reactivated motility was stable for at least 30 min at constant MgATP. These highly coupled models were used to show (1) that development of coordination in the ciliary tuft occurs at a higher substrate concentration range (10–25 μM) than that required to initiate motility per se (2–10 μM); (2) that outer dynein arms may not contribute to beat frequency at substrate concentrations below 35 μM; and (3) that vanadate has effects both on beat frequency and coordination of the tufts.
Fungal nuclear behavior analysed by ultraviolet microbeam irradiation
Authors: McKerracher Lisa J. , Heath I. Brent
Published: February 2005

During hyphal tip growth in the fungus Basidiobolus magnus, nuclei normally maintain a constant distance from the advancing cell apex by continuously migrating forward. It is not known whether the mechanism that produces nuclear movement also mediates nuclear positioning, or whether these two processes are under separate control. By irradiating small cytoplasmic regions with an ultraviolet microbeam, the coordination between movement and positioning could be disrupted. Regardless of the distance of the target from the nucleus, anterior irradiations (those ahead of the nucleus) caused the nucleus to stop or move backwards, whereas posterior (behind the nucleus) irradiations caused an acceleration in the nuclear velocity. The nucleus retained its ability to move following irradiation, so there was only loss of control over normal positioning. These results suggest that movement and positioning are mediated by different mechanisms. Quantitative microtubule analysis demonstrated that microtubules in the target region had been depolymerized, but in other regions of the cell they were apparently normal. We suggest that the depolymerization of microtubules affects nuclear movement by altering the tensile strength of the cytoplasm, and that cytoskeletal tension mediate nuclear positioning.

We also found that accelerated nuclear movement could occur when most of the microtubules surrounding the nucleus were depolymerized. A comparison of the microtubule population surrounding the nucleus in unirradiated versus irradiated cells suggested that microtubules move with nuclei. Therefore, the nucleus does not appear to move via a direct interaction with microtubules.
The use of submicroscopic gold particles combined with video contrast enhancement as a simple molecular probe for the living cell
Authors: De Brabander M. , Nuydens R. , Geuens G. , Moeremans M. , De Mey J.
Published: February 2005

We describe a new approach to probe the molecular biology of the living cell that uses small colloidal gold particles coupled to specific ligands. They are visualized in cells by bright‐field, video enhanced contrast microscopy. We describe the basic aspects of the technique and provide examples of applications to intracellular motility, cell membrane dynamics, receptor translocation, internalization, and intracellular routing. We also provide examples of the use of this approach in immunospecific labelling of cells and tissue sections.
Duplication of the flagellar apparatus and cytoskeletal microtubule system in the alga Polytomella
Authors: Aitchison William A. , Brown David L.
Published: February 2005

We have used immunofluorescence staining with monoclonal antibodies to tubulin and to components of the flagellar basal apparatus to examine duplication of the basal apparatus and the microtubule system assembled from it during cell division in the quadriflagellate alga Polytomella. The monoclonal antitubulin, prepared against Polytomella flagellar axonemes, detects Polytomella and mammalian tubulin by immunoblotting. By immunofluorescence and immunogold electron microscopy, it detects all microtubular structures that have been described in Polytomella. One of the antibodies generated using the isolated basal apparatus as immunogen appears to stain four of the eight basal body rootlets and is used in this study to detect early stages in the duplication of the flagellar apparatus. A cytoplasmic microtubule system is present, the elongate morphology of the cell is maintained, and the cells are motile throughout mitosis. The closed mitotic spindle forms perpendicular to the long axis of the cell. During mitosis, the newly formed basal bodies mature and four additional elongating flagella appear. Following mitosis, the eight flagella segregate into two groups, which begin to separate towards opposite poles of the cell. Concomitant with this separation, the rootlets of the parental basal apparatus separate and new rootlets are detected. We suggest that the components of the parental flagellar apparatus are segregated equally to the daughter cells. An interphase cytoskeletal microtubule array is assembled from each basal apparatus, and the morphology of the two cells is progressively established during cytokinesis.
Prelimlnary report on the ultrastructural organization of the contractile appendix of Tontonia appendiculariformis (ciliophora oligotrichina)
Authors: Greuet Claude , Gayol Philippe , Salvano Paule , Laval‐Peuto MichèLe
Published: February 2005

Tontonia appendiculariformis is a marine planktonic ciliate with a long tail. The tail can contract rapidly, becoming transformed into an oval mass one‐twentieth of its original length. The highly complex ulrastructure of the tail is described here in detail. A large part of the volume of the tail contains numerous more or less parallel membranous tubes. The membrane of the tubes has numerous invaginations and is probably derived from the smooth endoplasmic reticulum. This tubular material forms a continuous layer around the tail, interrupted in only one region, which contains cilia. Associated with the cilia are basal fibres with a periodically banded appearance. The tubular layer forms several folds separated by hyaloplasm containing many mitochondria. The pellicle of the tail is thrown into numerous pleats. It comprises a perilemma, a plasmalemma, and complex alveoli, but epiplasm and microtubules are absent. The alveoli appear to form septa within the folds of the layer of membranous tubes. In the region where the tail is attached to the body of the ciliate there are conspicuous bundles of microtubules and microfilaments. The membranous tubes and septa appear to be connected to small bundles of microfilaments, which presumably represent the contractile material. However, we consider the membranous tubes as potentially active in producing the change in shape. Although the structure of the tail of Tontonia is unique, there are certain similarities to the stalk of the Tintinnina and also to the motile extension of the dinoflagellate Erythropsidinium.
A model for the molecular basis of filament contractility and sliding as demonstrated by helix models
Authors: Jarosch Robert
Published: February 2005

The twisting behavior of α helices has hardly been considered hitherto with regard to the function of proteins. The well‐known electrostatic repulsion between the highly charged side chains, which depends on their interaction with ions, is absolutely connected with torsional rotations of the helix as long as its hydrogen bonds hold. This means a direct transformation of chemical into mechanical energy. However, the stability of a twisted single α helix with charged side chains is low in an aqueous environment. It may easily ball up to form a globular molecule with nonhelical regions of the polypeptide chain. This corresponds to a primitive contraction that obviously occurs with spasminlike proteins that contain strongly twisted filaments as Salisbury [J. Submicrosc. Cytol. 15:105–110, 1983] has shown. Steps that increase the stability and rigidity of α helical filaments are (1) the formation of coiled‐coils, (2) self‐intertwining (“telephone cord phenomenon”) or intertwining with other coiled‐coils as shown with the intermediate filaments, and (3) association with cytoskeletal elements (microfilaments, protofilaments of microtubules) that contain globular subunits. These coarser elements are rotated by winding and unwinding of the smaller helical molecules and thus transmit the torsion produced in the α helices to the microscopic level by the sliding (screwing) motion and the shearing effect that is connected with the waves of a rotating helix. Particles are transported if connected to the helical side arms. Since the displacement of the side arms seems to occur along the single protofilaments of a microtubule, a rotation of these protofiiaments is suggested. The bidirectional transport of particles along single microtubules may be explained by the association of left‐ and right‐handed helices with the protofilaments. According to the models, parallel and antiparallel sliding of neurofilaments and neurotubules is suggested.
Intracellular particle motions (cytoplasmic streaming) in staminal hairs of Setcreasea purpurea: Effect of azide and low temperature
Authors: Tucker Edward B. , Allen Nina Stromgren
Published: February 2005

Cytoplasmic streaming and its response to azide and low temperature were examined by using high‐resolution video‐enhanced light microscopy in Setcreasea purpurea staminal hair cells of immature flowers. Particles and organelles examined moved along well‐defined pathways, in repeated and unequal saltatory steps, at different rates and sometimes against the main direction of flow (bidirectionally) in both transvacuolar strand and peripheral cytoplasm. Particle movements were reversibly inhibited with azide. Low temperatures caused transvacuolar strands to shift or break. This cytoplasm accumulated in areas outside of the vacuole where spherosomes continued to saltate, but not along well‐defined pathways. In the peripheral cytoplasm, however, the spherosomes continued to move normally, amyloplasts became swollen, and they plus the other organelles (except spherosomes) were stationary. Normal particle movements were obtained when chilled cells were rewarmed to 27°C for ca 15 min.
Effects of colcemid and taxol on microtubules and intermediate filaments in chick embryo fibroblasts
Authors: Forry‐Schaudies Suzanne , Murray John M. , Toyama Yoshiro , Holtzer Howard
Published: February 2005

Reports on how changes in microtubule (MT) distribution or polymerization affect the distribution of intermediate filaments (IFs) differ. Therefore, we have used cytoimmunofluorescence techniques and electron microscopy to systematically examine and compare the arrangements of MTs and IFs in cultures of chick embryo fibroblasts under the following conditions: at different times during the cell cycle, in the presence of Colcemid or of taxol, in the presence of both drugs in succession or simultaneously in varying ratios, and during recovery from treatment with Colcemid or taxol.

We have found that depolymerization of MTs by 1 μM Colcemid resulted in the rapid formation of massive IF‐cables, structures distinct from “collapsed IFs” or “juxtanuclear coils.” Neither the rapid formation of IF‐cables nor their dispersion during recovery required protein synthesis. Cells treated with 10 μM taxol rapidly formed MT‐bundles, as well as aggregates of intertwining IFs, termed “IF‐skeins.” MT‐bundles and IF‐skeins displayed strikingly complementary distributions. This reciprocal distribution of packed MTs and IFs was also obvious in untreated anaphase and telophase cells. When 10 μM taxol and 1 μM Colcemid were applied simultaneously, the complementary distributions of MT‐bundles and IF‐skeins mimicked those in taxol alone. This ability of taxol to block Colcemid's effects was concentration dependent Decreasing the taxol:Colcemid ratio allowed the depolymerization of MTs, which correlated with the formation of IF‐cables.
Dynamic aspects of the contractile system in Physarum Plasmodium: II. Contractility of triton cell models in accordance with the contraction and relaxation phases of the plasmodia
Authors: Ishigami Mitsuo , Hatano Sadashi
Published: February 2005

The contractility of Physarum plasmodium was investigated using cell models that were prepared by treating thin‐spread plasmodia with ice‐cold 0.2% Triton X‐100. Cell models obtained from the anterior regions of the thin‐spread plasmodia in the contraction phase retained many birefringent cytoplasmic fibrils. The fibrils vigorously contracted on addition of ATP, inducing simultaneous contraction of the whole cell models. In contrast, cell models prepared from the anterior regions in the relaxation phase scarcely contained the birefringent fibrils and exhibited only weak contractility on addition of ATP. The posterior regions of the thinspread plasmodia, which were composed of ramified plasmodial strands, always retained many fibrils when treated with the Triton solution and showed intensive contraction on addition of ATP.

SDS‐polyacrylamide gel electrophoresis showed that the model was enriched for actin and myosin. About 40% of the actin was extracted from the plasmodium by the Triton treatment, while scarcely any myosin was extracted.

Fragmin, a F‐actin‐fragmenting factor, caused the birefringent fibrils to diminish in the presence of Ca2+, but more than 30 minutes was required for their complete disappearance. The birefringent fibrils weakened by 30‐minute fragmin treatment disappeared immediately on addition of ATP or AMP‐PNP.
Cytoskeletal architecture and motility in a giant freshwater amoeba, Reticulomyxa
Authors: Koonce Michael P. , Euteneuer Ursula , McDonald Kent L. , Menzel Diedrik , Schliwa Manfred
Published: February 2005

Reticulomyxa is a large, multinucleated freshwater protozoan with striking intracellular transport. Cyloplasmic streaming and saltatory movements of individual organelles (at rates of up to 25 μm/sec) are observed within the naked cell body and the extensive reticulate peripheral network of fine cytoplasmic strands. As demonstrated by video‐enhanced light microscopy, individual organelles move only when associated with cytoskeletal linear elements. The linear elements are composed of mixed colinear bundles of microtubules and actin filaments, which form the backbone of the reticulopodial network. The constant branching, sprouting, and fusion of network stands suggest unique membrane properties and an unusually dynamic cytoskeleton. The electrophoretic mobility of Reticulomyxa tubulins and the lack of crossreactivity with several antibodies known to react with many plant and animal tubulins suggest that they may differ from other tubulins more widely than might be expected. Reticulomyxa's large size, the rapidity and pervasiveness of the two forms of transport, and the simple and ordered cytoskeleton make the organism well suited for future studies on the mechanisms of intracellular transport.