bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of villin on the polymerization and subunit exchange of actin
Authors: Wang Yu‐Li , Bonder Edward M. , Mooseker Mark S. , Taylor D. Lansing
Published: February 2005

We have investigated the Ca2+‐dependent interactions of villin, a protein of the intestinal microvillar core, with actin by monitoring resonance energy tranfer between fluorescently labeled actin subunits. In the presence of elevated free Ca2+(∼20 μM), villin affects both the nucleation and the elongation phases of actin polymerization. Consistent with previous reports, villin stimulates the nucleation process and will form stable nuclei under depolymerization conditions. Compared to the control, the net rate of polymerization is slightly inhibited at low con‐centrations of villin (villin/actin ∼ 1:400) but is stimulated at higher concentrations (villin/actin > 1:100). Villin also significantly increases the critical concentration of actin polymerization. Addition of either villin or villin‐actin complexes induces depolymerization of preassembled actin filaments. This villin‐induced depolymerization is reversible upon removal of free Ca2+ or upon the addition of phalloidin. The exchange of actin subunits at steady state is inhibited at low concentrations of villin (villin/actin ∼ 1:200) but is stimulated at higher concentrations (villin/actin ∼ 1:50). None of the above effects is observed at < 10−8 M free [Ca2+].
Quantitative analysis of axonemal bends and twists in the quiescent state of ciona sperm flagella
Authors: Omoto C. K. , Brokaw C. J.
Published: February 2005

A simple planar model of sliding can predict the amount of sliding required to form a certain degree of bend. The accuracy of this prediction relies on the assumptions that no twists occur in the axoneme and that no sliding occurs at the base. However, previous studies indicated that twists may occur.

This paper explores a new method for quantitating and analyzing twists. Preliminary results using this method showed that there were twists. In order to control for possible artifacts due to fixation and other preparative procedures, the characteristic S‐shaped quiescent state of Ciona spermatozoa was studied.

Analyses of platinum replicas of those flagella in which this waveform is well preserved suggest that most, if not all, of the twists observed are due to the artifact of a curved shape settling onto a surface. Detailed analyses indicate that if twists do occur in quiescent sperm, they are probably less than 0.4 radian. Since axonemes are evidently easily twisted in rigor, and even after fixation, caution should be exercised in interpretation of axonemal twists.
Effects of folic acid upon filopodia of Dictyostelium discoideum vegetative amoebae
Authors: Rifkin Jared L. , Isik Ferda
Published: February 2005

Living vegetative D. discoideum amoebae were studied to determine whether their filopodia respond to folic acid, a chemoattractant for these cells. Exponentially growing amoebae (ca. 10 μm diameter) exhibit 5–30 μm long filopodia; at stationary phase, aggregation competent amoebae have numerous multibranched filopodia up to 100 μm long. Folic acid was observed to stimulate production, elongation, and branching of filopodia with its effects progressively changing as the amoebae approach aggregation. Filopodial construction was also found to be dependent upon Mg2+ levels. The significance of these results is discussed with respect to progressive changes within the vegetative phase as well as to the mechanisms of amoeboid movement, pseudopodial activity, and chemotaxis.
Light microscopic observations on the release of vesicles by isolated chromaffin cells
Authors: Edwards Charles , Englert David , Lotshaw David , Ye Han Zhang
Published: February 2005

Cultured bovine adrenal medullary chromaffin cells were stimulated with the secretogogues Ba2+ or carbamyl choline plus Ca2+. With video‐enhanced contrast, differential interference contrast microscopy, small vesicles were found to appear on the cell surface during stimulation. The structures were of lower refractive index than the cytoplasm, and their appearance required several tenths of a second. The vesicles are thought to correspond to omega figures seen with electron microscopy due to exocytosis. Many of the structures disappeared within a few seconds, but some appeared to coalesce into larger structures. The large structures may lead to the vacuoles that have been demonstrated to be present following stimulation. The nature of the cellular elements responsible for the vesicle which appeared on the surface was not found with either differential interference or interference reflection microscopy. The simplest explanation is that the refractive index of the elements is similar to that of the cell, and therefore the elements cannot be seen.
Microtubule crossbridging by Chlamydomonas dynein
Authors: Haimo Leah T. , Fenton Raymond D.
Published: February 2005

Dynein, obtained from axonemes of Chlamydomonas, binds by both its A and B ends to microtubules assembled from twice cycled (2 ×) and purified (6S) brain tubulin as well as to microtubules in native spindles, thereby inducing microtubule crossbridging. The two ends of the dynein arm exhibit distinct binding characteristics for the different microtubule preparations. Greater than 99% of the dynein arms are bound exclusively by their B ends to microtubules assembled from 6S tubulin in the presence of dynein and decorated to saturation. In contrast, greater than 80% of the dynein arms are bound by both their A and B ends to and, therefore, crossbridge 6S microtubules that are only partially dynein decorated. Binding of the A end of the dynein arm to saturated 6S microtubules can be enhanced by destabilizing the binding of the B end upon addition of ATP and vanadate. These observations suggest that Chlamydomonas dynein arms can bind by their A ends to microtubules assembled from 6S tubulin only when the B ends of the arms either are not bound or are bound but do not occupy all available dynein binding sites. Dynein exhibits a slight preference for binding by its A end to microtubules assembled from 2 × tubulin and containing microtubule associated proteins (MAPs). Approximately 90% of the dynein arms crossbridge adjacent 2 × microtubles that are only partially decorated. But as saturation of these microtubules with dynein is approached, the majority of the arms are bound solely by their A ends, while a smaller percentage are bound by their B ends or by both their A and B ends. These studies indicate that the type of microtubule as well as the degree of saturation of the microtubule with dynein can determine whether microtubule crossbridging occurs.
Time course of the motion of bull sperm flagella
Authors: Mulready Deborah E. , Rikmenspoel Robert
Published: February 2005

Detailed measurements were made of the time course of the motion of bull spermatozoa. Fourier analysis of the data showed the time course to be basically sinusoidal within 2% to 3%. An asymmetry in the motion was present, resulting in a second harmonic component in the Fourier spectra of normal sperm of approximately 11% of the main component. When the energy metabolism of the sperm was inhibited or when the external viscosity of the medium was raised, the asymmetry was reduced. When the internal Mg2+ content of the sperm was lowered, the asymmetry was increased. The asymmetries and the corresponding second harmonic components in the Fourier spectra were correlated with the overall bend shape of the sperm and with the curvature of the path in which the sperm were swimming. Model calculations showed that the asymmetry could reside in either the internal active moments in the sperms or in the stiffness of the sperm fiagella.
Automated methods for estimation of sperm flagellar bending parameters
Authors: Brokaw Charles J.
Published: February 2005

Parameters to describe flagellar bending patterns can be obtained by a microcomputer procedure that uses a set of parameters to synthesize model bending patterns, compares the model bending patterns with digitized and filtered data from flagellar photographs, and uses the Simplex method to vary the parameters until a solution with minimum root mean square differences between the model and the data is found. Parameters for Chlamydomonas bending patterns have been obtained from comparison of shear angle curves for the model and the data. To avoid the determination of the orientation of the basal end of the flagellum, which is required for calculation of shear angles, parameters for sperm flagella have been obtained by comparison of curves of curvature as a function of length for the model and for the data. A constant curvature model, modified from that originally used for Chlamydomonas flagella, has been used for obtaining parameters from sperm flagella, but the methods can be applied using other models for synthesizing the model bending patterns.
Reorientation of the golgi apparatus and the microtubule‐organizing center inside macrophages subjected to a chemotactic gradient
Authors: Nemere Ilka , Kupfer Abraham , Singer S. J.
Published: February 2005

Mouse peritoneal macrophages subjected to gradients of activated mouse serum were found by immunofluorescence observations to have their Golgi apparatus and their microtubule‐organizing center largely oriented in the direction of the gradient. By analogy with similar results obtained with motile fibroblasts, it is proposed that these two organelles are rapidly and coordinately reoriented inside the macrophages in order to direct the insertion of new membrane mass, via vesicles derived from the Golgi apparatus, into the leading edge of the cell. Consistent with the importance of such membrane insertion to cell migration, we found that the ionophore monensin, an inhibitor of Golgi functions, inhibited cell motility in the chemostactic gradient. It was further shown that several inhibitors of chemotaxis (monensin, cytochalasin D, cycloheximide) did not inhibit the reorientation of the Golgi apparatus/microtubule‐organizing center in cells exposed to a chemotactic gradient, and that the reorientation required extracellular Ca+2.
An EHNA‐senstive ATPase in unfertilized sea urchin eggs
Authors: Penningroth Stephen M. , Rose Patricia , Cheung Anne , Peterson Dolores D. , Rothacker Dana Q. , Bershak Patricia
Published: February 2005

In current purification strategies, affinity for microtubules or calmodulin is used to identify and purify cytoplasmic dynein‐like ATPase from cell‐free extracts of unfertilized sea urchin eggs. However, affinity purification procedures, though they define dynein‐like ATPase activity, have not yet proven to be quantitative. An alternative purification strategy capable of producing a high yield of enzyme would require a specific assay in order to monitor cytoplasmic dynein purity at each step.

In this study, we make a detailed comparison of the effects of EHNA on 22 different ATP‐metabolizing enzyme activities, including 13 Mg++‐ATPases. We isolate cytoplasmic dynein‐like ATPase activity from three species of sea urchin eggs and sperm and show by means of dose‐response curves that their sensitivities to inhibition by EHNA are very similar to one another. We demonstrate further that the EHNA dose‐response characteristics of fourteen other ATP‐metabolizing enzyme activities, including seven nondynein Mg++‐ATPases, differ quantitatively from those of dynein‐like ATPases.

In studies of three other agents (vanadate, Ca++/calmodulin, and Triton X‐100), we find that dynein‐like ATPases vary by two orders of magnitude in their sensitivities to inhibition by vanadate, and little or no stimulation by either Ca++/calmodulin or Triton X‐100 is seen. Our results suggest that inhibition by EHNA is a universal and specific property of dynein‐like ATPases, which ultimately should prove useful in the quantitative purification and characterization of cytoplasmic dynein‐like ATPase (s).
An investigation of the involvement of cytoskeletal structures and secretion in gliding motility of the marine diatom, Amphora coffeaeformis
Authors: Webster Daniel R. , Cooksey Keith E. , Rubin Robert W.
Published: February 2005

Gliding motility was investigated in the marine diatom, Amphora coffeaeformis. Ultrastructural, biochemical, and pharmacological protocols were employed to probe the possible involvement of cytoskeletal proteins and a secretory process in gliding motility. Motility rate was measured using a video recording apparatus, and the effects of various cytoskeleton‐disrupting drugs on motility were tested. Cytochalasins D and E, podophyllotoxin, and vinblastine (all at 25 μUg/ml) reversibly inhibited motility, as did monensin (10 μUM) and pronase (25 μUg/ml). Biochemical protein analysis of whole‐cell extracts by one‐ and two‐dimensional polyacrylamide gel electrophoresis revealed polypeptides comigrating with rabbit skeletal muscle actin and bovine brain tubulin; however, specific assays used to separate actin from whole‐cell preparations gave ambiguous results. Ultrastructural studies revealed the presence of extracellular material between the raphe canal and the substratum in motile cultures. An assay was devised for the detection of radioactively labeled material (MW > 1800 Daltons) released by motile cultures into the culture medium. When cultures were treated with either an anticytoskeletal drug or monensin, motility was inhibited while the amount of measured radioactivity increased over solvent‐treated control groups. The results from this study indicate possible roles for both actin‐ and tubulin‐based structures in gliding motility of Amphora. Though secretion may be necessary for gliding to occur, its exact relationship to motility was not deduced. The data obtained in this study are compatible with a theory for the mechanism of gliding which involves the surface translocation of externally exposed membrane proteins against an immobile matrix of substratum‐attached secreted material to generate the force required for movement.
Ionic control of locomotion and shape of epithelial cells: I. Role of calcium influx
Authors: Mittal Ajay Kumar , Bereiter‐Hahn Jürgen
Published: February 2005

The role of calcium in the induction of locomotion, control of direction of locomotion, and modulation of shape of epithelial cells derived from Xenopus laevis tadpole epidermis is investigated. Local influx of calcium is achieved by electrophoretic release of small amounts of calcium from a micropipette (tip diameter 0.1–0.5 μUm) closely apposed to the cell body or lamella. The cells are made permeable for calcium by calcium ionophore A23187, and they are kept in Ca++‐free, Mg++‐rich EGTA Ringer. Another method used to induce Ca++ influx is local application of A23187 while cells move in normal culture medium.

Influx of Ca++ into the lamella induces a localised increase in thickness and enlargement of the lamella. Stationary cells become active and show movement in the direction of the Ca++ gradient. Fried‐egg‐shaped cells tend to acquire a semicircular shape and start moving. Moving cells change the direction of their locomotion, following the direction of Ca++ release. Influx of Ca++ in the cell body region induces its contraction concomitant with an increase in lamellar area.

These observations suggest the presence of two different Ca++‐sensitive components: an actomyosin meshwork in the cell body and an actin gel in the lamella. Influx of Ca++ induces contraction of actomyosin and solation of actin gel. Interaction of these two systems would explain modulation of shape and generation of locomotion in epithelial cells.
High‐speed cinematographic analysis of the movement of Chlamydomonas
Authors: Rüffer U. , Nultsch W.
Published: February 2005

Using high‐speed microcinematography (100–500 f/s) the movement of Chlamydomonas reinhardtii mutant 622 E has been studied with frame‐by‐frame analysis. The stigma lies in the cell equator, displaced out of the flagellar plane anticlockwise by an angle of about 45°. During forward movement the cells rotate anticlockwise about their long axis with a frequency of 1.4–2 Hz (maximum 2.5 Hz). The rotation is caused by a lateral component of 3‐dimensional beating of the flagella during the effective strokes. The helical swimming path is a result of an unequal flagellar beating. This is normally synchronous, but synchrony is interrupted from time to time by an additional beat of the outward directed flagellum, in our study one after about every twenty beats on average. These results are discussed and compared with the results published by other groups.
A versatile and quantitative computer‐assisted photoelectronic technique used for the analysis of ciliary beat cycles
Authors: Sanderson Michael J. , Dirksen Ellen R.
Published: February 2005

The beat cycles of rabbit tracheal cilia in culture and Mytilus laterofrontal cirri were recorded using a phototransistor, transillumination, video, and phase‐contrast microscopy. The photoelectronic signal and video image of the ciliary activity were simultaneously recorded as a composite image. The photoelectronic signal was converted into a digital signal by a data acquisition system for further computer processing.

By the selection of a small detector area and accurate detector alignment, a simple, repetitive photoelectronic signal representing ciliary activity was obtained. This signal records the ciliary beat frequency and demonstrates the triphasic nature of the beat cycle. The photoelectronic signal can be precisely correlated with the ciliary activity by analysis of the composite video recordings to provide the duration of the effective, recovery, and rest phases of the beat cycle. The videophotoelectronic signal correlations were verified by high‐speed cinematography. High‐speed films of ciliary activity were digitized, and the image density of selected pixels was analyzed by computer with respect to time and ciliary motion.

These studies indicate that duration of the phages of the beat cycle are differentially reduced with increased beat frequency; the effective phase duration was quickly reduced to a minimum. This was followed by the reduction of the duration of the recovery phase to a minimum. The rest phase continues to be reduced without reaching a minimum, over the range of beat frequencies observed. These results suggest that ciliary beat frequency may be regulated either by modifying the rates of dynein‐microtubule interactions or the rate of transition from one beat phase to the next.
The antagonistic effects of 5‐hydroxytryptamine and methylxanthine on the gill cilia of mytilus edulis
Authors: Sanderson Michael J. , Dirksen Ellen R. , Satir Peter
Published: February 2005

The laterofrontal (LF) cirri on isolated gill filaments of Mytilus edulis, prepared in natural seawater, are active and initially beat with an average frequency of about 8 Hz (with a range of 6–14 Hz). However, the lateral (L) cilia on these filaments are arrested in a position at the end of their recovery stroke. Perfusion of the filament with artificial seawater (ASW), with or without 1% ethanol, has little or no biological effect on the activity of the LF cirri, although a transitory decrease in frequency often accompanies the perfusion process. The L cilia remain arrested during perfusion with ASW. The exposure of the gill to low levels of 5‐hydroxytryptamine (5HT) (10−8 < 5HT < 10−7 M) has no effect on the activity of the LF cirri but stimulates the L cilia to beat. Exposure to higher concentrations of 5HT (> 10−7 M) elevates the beat frequency of the L cilia and simultaneously inhibits the activity of the LF cirri, leading to their arrest in a position at the end of the effective stroke. This arrest of the LF cirri occurs as the L cilia attain a 5HT‐induced beat frequency between 12 to 14 Hz. The influence of 5HT on the L cilia and the LF cirri can be reversibly mimicked or enhanced by the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX). A concentration of 0.5 mM IBMX mimics low 5HT concentrations (about 10−7 M) by stimulating the L cilia to beat without affecting the beat frequency of the LF cirri. A combimation of 10−7 M 5HT and 0.5 mM IBMX in ASW mimics high (> 10−6 M) 5HT concentrations by arresting the LF cirri and increasing the beat frequency of the L cilia. Under these conditions, the threshold of the LF cirri arrest response is again found to occur as the L cilia attain a beat frequency of 12 – 14Hz. These results suggest that the mechanisms of LF cirri arrest and L cilia activation are mediated by 5HT ‐induced changes in intracellular cyclic AMP levels.
Localization of actin in Chlamydomonas using antiactin and NBD‐phallacidin
Authors: Detmers Patricia A. , Carboni Joan M. , Condeelis John
Published: February 2005

We have localized actin in gametes of Chlamydomonas reinhardi by two approaches: (1) indirect immunofluorescence with an affinity‐purified antibody and (2) staining with NBD‐phallacidin, a fluorescent reagent that binds only to F‐actin [Barak et al, 1980, Proc Natl Acad Sci, 77:980–984]. Staining of either mating type “plus” (mt+) or “minus” (mt−) gametes with antiactin antibody resulted in similar fluorescent images: most of the actin was located peripherally along the lateral and posterior aspects of the cells. There was diffuse staining centrally, but the flagella did not stain. No brightly stained spot was observed near the mt+ mating structure, the site where the fertilization tubule elongates with concomitant polymerization of actin [Detmers et al, 1983, J Cell Biol, 97:522–532]. Gametes stained prior to mating with NBD‐phallacidin showed no fluorescence above background, indicating that there were no concentrations of F‐actin in these cells. This suggested that the cytoplasmic staining observed with antiactin represented primarily a nonfilamentous form of the protein. In mating gametes staining with NBD‐phallacidin was detected only in the fertilization tubule, indicating that this was the only dense accumulation of filamentous actin within the cells. Mating gametes stained with antiactin antibody exhibited cytoplasmic fluorescence that was slightly more punctate than prior to mating, and the fertilization tubule was brightly stained. Our observations suggest that the site‐specific polymerization of actin within the fertilization tubule occurs in the absence of a concentrated pool of actin subjacent to the mating structure.
Alpha actinin distribution and extracellular matrix products during somitogenesis and neurulation in the chick embryo
Authors: Lash James W. , Ostrovsky David , Mittal Balraj , Sanger Joseph W.
Published: February 2005

A discrete stage in two different morphogenetic processes has been examined employing fluorescently labelled alpha‐actinin as a probe to localize native alpha‐actinin and antibodies to localize fibronectin and collagen type I. The stage of somitogenesis examined is the transition from the compact mesenchymal somitic mass to the epithelial somitic vesicle (ie, epithelialization of the somite). The stage of neurulation examined is the transition from the relatively flat neuroepithelium to the approximation of the neural folds. Before these morphogenetic movements begin, the neuroepithelium is sitting upon a basal lamina and interstitial collagen, and the somite is surrounded by a meshwork of interstitial collagen. During both of these processes, the cells become narrowed at their apices in the region of the tissue that is becoming concave, and alpha‐actinin is localized in the apices. The localization of intracellular alpha‐actinin and extracellular fibronectin, and the distribution of collagen, suggest that there is a coordinated appearance and distribution of these molecules that is temporally associated with these discrete morphogenetic events.
Regulation of sperm flagellar movement by protein phosphorylation and dephosphorylation
Authors: Murofushi Hiromu , Ishiguro Koichi , Takahashi Daisuke , Ikeda Jun , Sakai Hikoichi
Published: February 2005

Flagellar motility of Triton models of sea urchin spermatozoa was reactivated by cyclic AMP‐dependent protein kinase and a protein factor, termed motility activator, both of which were prepared from the detergent‐extract of sea urchin spermatozoa. It was shown that phosphorylation of the motility activator by the protein kinase is necessary for the reactivation of flagellar motility [Ishiguro et al, J. Cell Biol. 92:777–782, 1982; Murofushi et al, in “Biological Functions of Microtubules and Related Structures,” Academic Press, 1982]. Reactivating factor was also detected in a KCI‐extract of the axoneme fraction devoid of the detergent‐extractable materials. The activity of this factor was also cyclic AMP‐ and protein kinase‐dependent. Furthermore, when freshly prepared Triton models were treated with phosphoprotein phosphatase prepared from bovine cardiac muscle, the flagellar motility was drastically suppressed. This inhibition of the motility was partially recovered by the addition of cyclic AMP and protein kinase to the phosphatase‐treated models.
Incorporation and turnover of labeled exogenous tubulin in the mitotic spindles of Chaetopterus oocytes and HeLa cells
Authors: Goode Dennis , Sarma Vidya
Published: February 2005

The incorporation of tubulin into mitotic spindles in situ was studied by incubating permeabilized mitotic cells in solutions containing [3H]GTP‐labeled or dichlorotriazinylamino fluorescein (DTAF)‐labeled tubulin. Metaphase HeLa cells or spindle‐containing “minicells” from Chaetopterus oocytes were lysed in a microtubule‐assembly buffer plus 0.5% Nonidet P‐40, 1 mg/ml 120,000g supernatant mammalian brain tubulin, and [3H]GTP. After different periods of incubation, mitotic spindles were isolated in 2 M‐glycerol‐containing assembly buffer and separated from unbound counts by centrifugation through a 4 M‐glycerol cushion; 3H counts per mg protein increase linearly for 8–12 min and then reach a plateau or steady state in both Chaetopterus oocytes and HeLa cells. Addition of 4 mM CaCl2 blocks or reverses incorporation. Little or no [3H]GTP is incorporated if exogenous tubulin or lysed cells are omitted from the assembly mixture.

To measure the loss rate of [3H]GTP‐tubulin from mitotic spindles, cells were incubated in tubulin plus [3H]GTP for 30 min, and a 20‐fold excess of cold GTP (2 mM) was added. Samples were removed after incubation for different periods, and spindles were isolated as described above and counted for 3H content. [3H]GTP is lost from spindles at a rate of about 16%/min until a new steady state is reached in about 8 min. These results are consistent with an incorporation and turnover of [3H]GTP‐tubulin in spindle microtubules of these lysed‐cell models.

The location of this newly incorporated tubulin in the spindle was investigated by incorporating fluorescent DTAF‐tubulin into mitotic spindles of these lysed cell types. A short pulse (2–5 min) appears to label microtubules (MTs) near metaphase chromosomes and longer exposures label the entire spindle.

The rates of incorporation and turnover that we see by [3H]GTP and fluorescent tubulin incorporation in situ are faster than those observed with brain MTs at steady state in vitro but are in the range of the rates of spindle fiber formation in prophase, and spindle MT reassembly after cooling.
Motility and centrosomal organization during sea urchin and mouse fertilization
Authors: Schatten Heide , Schatten Gerald
Published: February 2005

Motility and the behavior and inheritance of centrosomes are investigated during mouse and sea urchin fertilization. Sperm incorporation in sea urchins requires microfilament activity in both sperm and eggs as tested with Latrunculin A, a novel inhibitor of microfilament assembly. In contrast the mouse spermhead is incorporated in the presence of microfilament inhibitors indicating an absence of microfilament activity at this stage. Pronuclear apposition is arrested by microfilament inhibitors in fertilized mouse oocytes. The migrations of the sperm and egg nuclei during sea urchin fertilization are dependent on microtubules organized into a radial monastral array, the sperm aster. Microtubule activity is also required during pronuclear apposition in the mouse egg, but they are organized by numerous egg cytoplasmic sites. By the use of an autoimmune antibody to centrosomal material, centrosomes are detected in sea urchin sperm but not in unfertilized eggs. The sea urchin centrosome expands and duplicates during first interphase and condenses to form the mitotic poles during division. Remarkably mouse sperm do not appear to have the centrosomal antigen and instead centrosomes are found in the unfertilized oocyte. These results indicate that both microfilaments and microtubules are required for the successful completion of fertilization in both sea urchins and mice, but at different stages. Furthermore they demonstrate that centrosomes are contributed by the sperm during sea urchin fertilization, but they might be maternally inherited in mammals.
Cell shape and organization of F‐actin and microtubules in randomly moving and stationary amebae of Dictyostelium discoideum
Authors: Roos U.‐P. , De Brabander M. , Nuydens R.
Published: February 2005

We investigated, by video‐light microscopy and fluorescence microscopy with probes specific for microtribules (MTs) and F‐actin, the relationship between cytoskeletal elements, cell shape and behavior of vegetative, undifferentiated amebae of Dictyostelium discoideum, strain NC‐4. In an unconstrained situation, as on the underside of a coverglass in a thin layer of liquid medium, interphase cells moved around randomly in a polypodial or monopodial fashion. Locomotion was characterized by the formation of pseudodigits, rounded or pointed pseudopodia, and retraction fibers. F‐actin occurred in all these structures, as well as in a thin cortical layer. Microtubules extended into some of the cellular extensions rich in F‐actin. Pseudopodial activity, but not locomotion, also took place at the interface between medium and air, demonstrating that ameboid movement requires contact with a solid substrate. Stationary mitotic amebae on glass were studded with continuously changing, peripheral spike‐shaped filopodia that also contained F‐actin. During telophase and cytokinesis the spikes were gradually replaced by pseudopodia in transition to the fully motile phase.

In live cells, the nucleus‐associated body (NAB), which is at the center of the complex of cytoplasmic MTs [CMTC; term from Brinkley, Fuller, and Highfield, 1975] was in a rather fixed position; it did not orient in a concerted fashion to follow changes in the direction of cell movement. In amebae fixed and processed for fluorescence microscopy after a period of recorded movement, the NAB was not preferentially positioned with respect to the nucleus and the direction of movement. It is unlikely that the NAB exerts a directional control during randon ameboid movement. The complex of cytoskeletal MTs must be very dynamic or flexible to adjust to the rapid changes of cell shape.
Rearrangement of tubulin, actin, and myosin in cultured ventricular cardiomyocytes of the adult rat
Authors: Guo Juan‐Xia , Jacobson Stuart L. , Brown David L.
Published: February 2005

Antitubulin, phalloidin. and antimyosin were used to study the distribution of microtubules, microfilaments, and myofibrils in cultured adult cardiomyocytes. These cells undergo a stereotypic sequence of morphological change in which myotypic features are lost and then reconstructed during a period of polymorphic growth. Microtubules, though rearranged during these events in culture, are always present in an organized network. Myosin and actin structures, on the other hand, initially degenerate. This initial degeneration is reversed when a cell attaches to the culture substratum. Upon attachment, new microtubules are laid down as a cortical network adjacent to the sarcolemma and, subsequently, as a network in the basal part of the cell. Actin and then myosin filament bundles appear next, in a pattern corresponding to the pattern of the microtubules. Finally, striated myofibrils are formed, first in the central part of the cell, and subsequently in the outgrowing processes of the cell, A mechanism is suggested by which the eventual polymorphic shape of a cell is related to the shape of its initial area of contact with the culture substratum. Finally, a model of myofibrillogenesis is proposed in which microtubules participate in the insertion of myosin among previously formed actin filament bundles to produce myofibrils.
Naturally occurring tubulin‐containing paracrystals in Allogromia: Immunocytochemical identification and functional significance
Authors: Rupp Gerald , Bowser Samuel S. , Mannella Carmen A. , Rieder Conly L.
Published: February 2005

Bundles of microtubules (MTs) are readily visualized in vivo by videomicroscopy in highly flattened reticulopodia of the foraminiferan protozoan Allogromia sp. strain NF. In this report we use videomicroscopy, immunocytochemistry, and high‐voltage electron microscopy to characterize the dynamic changes that occur in this extensive MT cytoskeleton, and in the associated cytoplasmic transport, during induced withdrawal and subsequent reextension of reticulopodia. Within seconds after application of the withdrawal stimulus (seawater substitute made hypertonic with MgCl2) intracellular bidirectional transport along linear MT‐containing fibrils ceases and is replaced by an inward, constant‐velocity flow of cytoplasm along the fibrils. As withdrawal continues, most fibrils become wavy and coalesce to form phase‐dense pools. These wavy fibrils and phase‐dense pools contain a paracrystalline material and few if any MTs. Same‐section correlative immunofluorescence and high‐voltage electron microscopy reveal that the paracrystalline material contains tubulin. During recovery linear fibrils (MTs) rapidly extend from the phase‐dense pools (paracrystals), which concurrently shrink in size, thus reestablishing normal network morphology and motility. We conclude that the MT cytoskeleton in Allogromia reticulopodia is transfonned during withdrawal into a tubulin‐containing paracrystal, which serves as a temporary reservoir of MT protein and an initiation site for MT regrowth.
Relationship between intermediate filaments and microfilaments in cultured fibroblasts: Evidence for common foci during cell spreading
Authors: Green Kathleen J. , Talian John C. , Goldman Robert D.
Published: February 2005

Spreading and fully spread chick embryo fibroblasts (CEF) were examined by double‐label fluorescence microscopy using the actin‐specific probe rhodamine‐phalloidin and an antibody directed against CEF intermediate filaments (IF). During midspreading, a striking relationship became discernible: statistical analysis showed that approximately half of the cell population exhibited one or more phase‐dense, phalloidin‐binding nodules that appeared to act as foci from which IF diverged. Coincidence between actin‐containing structures and IF was not limited to these centers; IF could also frequently be seen running in close parallel arrays with stress fibers.

Ultrastructural analysis confirmed the presence of non‐membrane‐bound out‐pocketings along the length of stress fibers from which 10‐nm IF diverged. These structures varied in size and shape, and displayed a dense, fine fibrillar appearance. IF and microfilaments (MF) were distinguished by size and by decoration of MF with myosin subfragment‐1. Other IF‐MF interactions were seen in cells of all stages: IF were observed to loop through stress fibers, most frequently at the cell margins. In colchicine‐treated cells, IF became redistributed into cables that often ran parallel and appeared to merge with stress fibers. Cytochalasin D‐treated CEF exhibited loose aggregates of actin‐containing material that appeared to be associated with IF.

These results suggest the possibility of an interaction between actin‐containing structures and IF, particularly during cell spreading in cultured fibroblasts.
Effects of pteridines on the filopodia of Dictyotelium discoideum vegetative amoebae
Authors: Rifkin Jared L. , Wali Abdul W.
Published: February 2005

Living vegetative amoebae of NC‐4H Dictyostelium discoideum were studied to determine if a variety of pteridines had any effect on the filopodia. We observed that production, elongation, and branching of these filopodia were stimulated by pteridines that are chemoattractants for cells of this strain. This stimulation occurs at chemotactically effective concentrations and is observed before motility is evident. A relationship between filopodia and chemoattractant signal processing is discussed.
Bivalent orientation and behavior in crane‐fly spermatocytes recovering from cold exposure
Authors: Janicke Marie A. , LaFountain James R.
Published: February 2005

At metaphase in crane‐fly primary spermatocytes, the two sister kinetochores at the centromere of each homologue in a bivalent normally are adjacent and face the same pole; one homologue has all its kinetochore microtubules (kMTs) extending toward one pole and its partner has all its kMTs extending toward the opposite pole. In contrast, during recovery from exposure to 2°C, one or both homologues in many metaphase bivalents had bipolar malorientations: all kMTs of one kinetochore extended toward one pole and some or all those of its sister extended toward the other. Metaphase sister kinetochores that had most of their kMTs extending toward the same pole were adjacent, and those with most extending toward opposite poles were separated from each other. Distances between homologous centromeres were similar to those in properly oriented bivalents. Maloriented bivalents were tilted relative to the spindle axis, and analysis of living cells showed that tilted configurations were rare during prometaphase in untreated cells but frequently arose in cold‐recovering cells as initial configurations, then persisted through metaphase. This was in contrast to unipolar configurations of bivalents (configurations suggesting orientation of both homologous centromeres toward the same pole), which always reoriented shortly after the configuration arose. We conclude that in cold‐recovering cells, bipolar malorientations are more stable than unipolar malorientations, and the orientation process is affected such that bipolar malorientations arise in bivalents upon initial interaction with the spindle and persist through metaphase.
Alpha‐actinin accumulation in the cortex of echinoderm eggs during fertilization
Authors: Hamaguchi Yukihisa , Mabuchi Issei
Published: February 2005

Redistribution of alpha‐actinin during fertilization was investigated by means of the microinjection of fluorescently labeled egg alpha‐actinin in the sea urchin, Hemicentrotus pulcherrimus. Upon fertilization, labeled alpha‐actinin accumulated locally around the sperm binding site, where the fertilization cone formed soon afterwards. The accumulation propagated all over the cortex within 10 sec after a latent period of 10–20 sec. When an egg in Na‐free seawater was injected with both alpha‐actinin and calcium buffer (intracellular free Ca2+ concentration = 9 μM), the accumulation of alpha‐actinin was similar to that in normal seawater, which suggests that the accumulation did not depend on the increase in intracellular pH but only on the increase in the intracellular free Ca2+ concentration. In immature oocytes the accumulation was detected in the cortical region, including the huge protruding cytoplasm where the sperm entered. When labeled egg alpha‐actinin was injected into starfish (Asterias amurensis) oocytes followed by insemination, it accumulated in the cortical layer in a manner similar to the case of sea urchin, except that the accumulation in fertilization cones of maturing oocytcs or reception cones of immature oocytes appeared ringlike and rodlike, respectively. Moreover, just after the arrival of the meiotic apparatus, egg alpha‐actinin accumulated in the cortical region, where the formation of the polar body was expected. This suggests that the meiotic apparatus somehow induced the differentiation of the cortex so as to form a polar body. It is concluded that the cortical region where alpha‐actinin accumulated coincided with the microfilament‐rich region. This suggests that alpha‐actinin plays a role in forming the cortical meshwork of actin filaments.
Is the hemidesmosome a half desmosome? An immunological comparison of mammalian desmosomes and hemidesmosomes
Authors: Jones Jonathan C. R. , Yokoo Karen M. , Goldman R. D.
Published: February 2005

Although the mammalian epidermal basal cell hemidesmosome bears some superficial resemblance to one half of a desmosome at the ultrastructural level, examination of the structure of the electron‐dense submembranous plaques of the hemidesmosome and desmosome reveals that they differ with respect to their overall morphology and dimensions. Based on these findings, we wondered whether components of the desmosome are present in the hemidesmosome. In order to determine this we prepared a number of stratified squamous epithelial tissues for indirect immunofluorescence using antibody preparations directed against known desmosome components including desmoplakin and certain glycoproteins. These antibody preparations do not show reaction with hemidesmosomes by indirect immunofluorescence criteria. We have also utilized bullous pemphigoid (BP) autoantibodies that have been shown to recognize hemidesmosomes in mammalian skin cells [Mutasim et al., J. Invest. Derm., 84:47–53, 1985]. Double label indirect immunofluorescence observations of neonatal mouse skin prepared using desmoplakin antibodies and BP autoantibodies reveal that hemidesmosomes that are stained by the BP autoantibodies are not recognized by the desmoplakin antibodies. We confirmed these findings at the ultrastructural level by indirect immunogold localization of desmoplakin antibodies and BP autoantibodies. Therefore, the hemidesmosome does not appear to be one half of a desmosome and may possess a very different molecular organization relative to the desmosome. We raise the possibility that the variability between the hemidesmosome and desmosome that we detect at the morphological and immunological level may reflect the functional differences of these two types of junctions.
Decoration of microtubules by fluorescently labeled microtubule‐associated protein 2 (MAP2) does not interfere with their spatial organization and progress through mitosis in living fibroblasts
Authors: Vandenbunder Bernard , Borisy Gary G.
Published: February 2005

Microtubule‐associated protein 2 (MAP2) derivatized with iodoacetamidotetramethylrhodamine or with iodoacetamidofluorescein binds to microtubules after injection into living interphase cells [Scherson et al, 1984]. The binding of derivatized MAP2 stabilized microtubules in vitro; it was therefore important to check if the binding of MAP2 in vivo perturbed the dynamics and organization of the microtubule network. We have addressed these questions by studying the effect of the injection of derivatized MAP2 on mitosis in PtK 1 cells and on the recovery of the microtubule network from low temperature incubation in interphase cells. We found that the presence of derivatized MAP2 did not change the duration of any mitotic stage and that the injected cell normally completed mitosis. We subsequently showed that the injected MAP2 bound to the microtubules within 5 minutes after injection and remained bound throughout the course of mitosis. The reorganization of the microtubule network upon cooling and rewarming was studied in the cytoplasm of human foreskin fibroblasts (356 cells). During the recovery, the distribution of the fluorescent MAP2 in living cells was identical with the microtubule pattern visualized by immunofluorescence in lysed and fixed cells.

In these experiments, the fluorescent MAP2 bound to microtubules can be considered as a nonperturbing reporter of the microtubule network. This result is discussed in terms of the role of MAPs in the dynamics and organization of microtubules in living cells.
Cyclical bending of two outer‐doublet microtubules in frayed axonemes of Chlamydomonas
Authors: Kamiya Ritsu , Okagaki Tsuyoshi
Published: February 2005

When detergent‐extracted cell models of Chlamydomonas reinhardtii were left in the presence of 1 mM Mg‐ATP for more than 30 minutes flagellar axonemes tended to become frayed into fine bundles of microtubules. Under such conditions, bundles made up of a pair of outer‐doublet microtubules displayed oscillatory bending movements of low (< 2 Hz) frequencies. The two doublet microtubules underwent association‐dissociation cycles coupled with gross bending movement. A model is presented to explain this phenomenon by unidirectional sliding interaction between the two microtubules.
Evidence for functional differences between two flagellar dynein ATPases
Authors: Penningroth Stephen M. , Peterson Dolores D.
Published: February 2005

Energy coupling in flagellar motility was investigated using demembranated, reactivated sea urchin spermatozoa (Arbacia punctulata). The ATP‐dependence of ATPase activity was investigated for ATP concentrations ranging from 4 μM to 600 μM ATP. Using Eadie‐Scatchard plot analysis, we identified two axonemal dynein ATPase activities. Their apparent Michaelis constants were calculated to be equal to 4 μM and 161 μM ATP, and they were referred to, respectively, as the high‐affinity dynein ATPase (HADA) and the low‐affinity dynein ATPase (LADA). Investigation of movement‐coupled ATPase activity (difference between the ATPase activities of reactivated and broken, immotile spermatozoa) indicated that HADA and LADA were both 65% movement‐coupled. The apparent Michaelis constants of movement‐coupled HADA and LADA, 12 μM and 271 μM ATP, respectively, were two‐ to four‐fold greater than the apparent Michaelis constants of movement‐uncoupled HADA and LADA. The apparent Michaelis constants for force generation and beat frequency of reactivated spermatozoa were determined to be 24 μM and 290 μM ATP, respectively. These results raise the possibility that flagellar force generation is controlled primarily by movement‐coupled HADA, and that flagellar beat frequency is controlled primarily by movement‐coupled LADA. Thus, mechanochemical activity in flagellar motility may be divided between two enzymatically and functionally distinct classes of flagellar dyneins.