bluuebunny/crossref_metadata_2025_split huggingface dataset:
Polymerization of additional actin is not required for capping of surface antigens in B‐lymphocytes
Authors: Jackman W. T. , Burridge K. 
Published: February 2005

AbstractCH12 is a murine B‐cell lymphoma whose surface immunoglobulin (sIg) and concanavalin A (Con A) receptors patch and cap readily. Actin may be involved in CH12 patching and capping, since fodrin and F‐actin collect under the cap, and cytochalasin D inhibits sIg capping. We have examined the state of the actin cytoskeleton during patching and capping. A wide range of concentrations of rabbit anti‐mouse antibody (RAM) and Con A were used to patch or cap CH12 cells. G‐actin was quantitated by DNase I inhibition, F‐actin was quantitated by fluorescence‐activated cell sorter analysis of fluorescent phalloidin staining, and actin nucleation sites were measured by pyrene actin polymerization. None of these methods detected any significant changes in actin when compared to control cells or untreated cells, leading us to conclude that increased actin polymerization is not necessary for capping to occur. The significance of these data to the membrane flow and cytoskeletal models of capping is discussed.
Control of reactivation and microtubule sliding by calcium, strontium, and barium in detergent‐extracted macrocilia of Beroë
Authors: Tamm Sidney L. 
Published: February 2005

AbstractMacrocilia of the ctenophore Beroë are activated to beat continuously in the normal direction by membrane‐mediated Ca2+ influx (Tamm: Journal of Comparative Physiology [A] 163:23–31, 1988a). Using saponin or Brij‐58 permeabilized models of macrocilia, we show that ATP‐reactivation of beating requires μM levels of free Ca2+, Ba2+, or Sr2+. Isolated macrocilia beat initially in reactivation solution (RS) containing Ca2+, Ba2+, or Sr2+ and then undergo microtubule sliding disintegration without added proteases. Addition of protease inhibitors to RS + 10−5 M Ca2+ prevents sliding disruption. Pretreatment in wash solution (containing 1 mM EGTA) without protease inhibitors, followed by RS + 10−5 M Ca2+ with protease inhibitors results in extensive sliding disintegration. However, treatment in wash solution followed by RS + protease inhibitors does not induce sliding. Therefore, Ca2+ is not required for proteolysis by endogenous proteases, but is necessary for sliding disintegration.Local iontophoretic application of Ca2+, Ba2+, or Sr2+ to permeabilized macrocilia in RS lacking these cations triggers motility and/or sliding disintegration. Extrusion of microtubules occurs from the tip or the base, depending on whether or not the macrocilium remains attached to its large actin bundle. Thin sheets of microtubules telescope out initially, due to synchronized sliding of subsets of doublet microtubules from parallel rows of axonemes.Macrocilia are one of the first examples of ATP‐induced microtubule sliding which retains Ca2+ sensitivity. In addition, the finding that Ba2+ and Sr2+ also trigger active sliding provides an additional method for investigating the control of dynein‐powered microtubule movements.
Cellular morphogenesis and the formation of marginal bands in amphibian splenic erythroblasts
Authors: Ginsburg Mary F. , Twersky Laura H. , Cohen William D. 
Published: February 2005

AbstractThe spleen of Ambystoma mexicanum (axolotl) larvae develops as a closed sac containing differentiating nucleated erythrocytes, and is typically isolated from the general circulation for about 10 days post‐hatching. Beginning 3–4 days posthatching, it can be removed intact for examination of the morphology and cytoskeletal structure of the erythropoietic cells. In the smallest (earliest) spleens, spheroidal cells predominate, while older ones contain a preponderance of cells exhibiting the flattened elliptical morphology typical of all non‐mammalian vertebrate erythrocytes. Most striking in the splenic erythroid population are cells with singly or doubly pointed morphology. Though common in the developing spleen and circulation of young larvae, pointed cells are less frequently encountered in the circulation of older larvae, indicating that they are intermediate stages in the differentiation of spheroids to flattened ellipsoids. This is supported by structural observations on cytoskeletons prepared from the splenic cells. Incomplete singly and doubly pointed marginal bands of microtubules are observed, many of which contain a pair of centrioles within or close to a pointed end, suggestive of organizing center function. The observations are consistent with a sequence of changes in cell morphology from spherical to doubly pointed to singly pointed to flattened ellipse, causally linked to stages of marginal band biogenesis.
Differential localisation of tyrosinated, detyrosinated, and acetylated α‐tubulins in neurites and growth cones of dorsal root ganglion neurons
Authors: Robson Susanne J. , Burgoyne Robert D. 
Published: February 2005

AbstractThe comparative distribution of tyrosinated, detyrosinated, and acetylated α‐tubulins was examined in neurites of rat dorsal root ganglion neurones in culture using immunofluorescence microscopy. Phase contrast observations of single neurones revealed that the neurites were actively motile, and rhodamine phalloidin staining of actin filaments showed the extent of lamellopodia and microspike projections from the growth cones. From double‐labelling experiments using antibodies against tyrosinated, detryrosinated, or acetylated α‐tubulin, it was found that the three different isoforms were differentially localised in neurites and growth cones. Detyrosinated and acetylated forms of α‐tubulin were in the main restricted to the neurites extending no further than the base of the growth cones. Tyrosinated α‐tubulin was, however, distributed throughout the body of the growth cone and into the base of some microspikes. Following treatment with taxol to promote microtubule assembly, detyrosinated and acetylated α‐tubulins were found to be colocalised with tyrosinated α‐tubulins throughout the growth cones of all cells examined. These results would be consistent with axonal transport of tyrosinated α‐tubulin followed by assembly in the growth cone and subsequent detyrosination and acetylation. In addition the presence of unmodified α‐tubulin in the growth cone may be necessary for the provision of labile microtubules for growth cone motility and extension.
Cytoskeletal features of the syncytial epidermis of the tapeworm Hymenolepis diminuta
Authors: Holy Jon M. , Oaks John A. 
Published: February 2005

AbstractA hallmark feature of parasitic platyhelminths is a cytoarchitecturally unusual syncytial epidermis composed of a peripheral layer of continuous cytoplasm (the ectocytoplasm) connected to underlying nucleated cell bodies by small cytoplasmic bridges. The helminth epidermis, or tegument, plays important roles in protection and nutrient acquisition; cestodes, in fact, completely lack a gastrointestinal tract and absorb all nutritive material through the tegument. Perhaps not surprisingly, the cestode tegument bears certain resemblances to the mucosal epithelium of the vertebrate small intestine, including the possession of a microvillous brush border upon the surface of the ectocytoplasm. In contrast to the intestinal epithelial cell, however, very little is known concerning the nature and organization of the cytoskeleton within the helminth epidermis. Therefore, a number of different microscopical preparative techniques were used to examine the tegument of the tapeworm Hymenolepis diminuta for the presence and distribution of microfilaments, intermediate filaments, and microtubules. It was found that both actin‐containing microfilaments and intermediate‐sized filaments are present but are restricted to specific locations along the plasmalemmae of the ectocytoplasm. In contrast, microtubules are found throughout the tegument, and are concentrated in the supranuclear regions of the perikarya and in the cytoplasmic bridges interconnecting the perikarya and ectocytoplasm. Unlike brush borders of most other epithelia, the cestode epidermal brush border lacks a filamentous terminal web and is instead associated with microtubules. A network of fine filaments, 5–8 nm in diameter but distinct from actin‐containing microfilaments, runs throughout the ectocytoplasm and appears to interlink tegumental vesicles. These fine filaments may represent the primary “skeletal” system responsible for maintaining the structure of the tegumental cytoplasm.
Acrylamide sensitization of the heat response of the cytoskeleton and cytotoxicity in attaching and well‐spread synchronous chinese hamster ovary cells
Authors: Wachsberger Phyllis R. , Coss Ronald A. 
Published: February 2005

AbstractThe vimentin intermediate filament (VIMF) network is more sensitive to heat‐induced disruption than either the microtubule (MT) or microfilament (MF) cytoskeletal (CSK) arrays in G1 Chinese hamster ovary (CHO) cells (Coss and Wachsberger: Radiation Research, 1987). We therefore investigated the effect of the VIMF disruptive agent, acrylamide (Eckert: European Journal of Cell Biology 37:169–174, 1985), on the heat response of synchronous CHO cells. Cells, either in the process of spreading (G1 or S phase) or in the well‐spread state (S phase), were exposed to a nontoxic concentration of 5 mM acrylamide, heated, and processed for immunofluorescence microscopy 30 min or 20 hr following the heat shock. Recovery from CSK disruption was related to cell survival.CHO cells, either in the process of spreading or in the well‐spread state, were sensitized to heat‐induced CSK disruption and cytotoxicity by acrylamide. Recovery from CSK disruption correlated with surviving fractions of cells treated in the G1 phase but not with surviving fractions of cells treated in the S phase and was independent of the degree of cell spreading. This correlation suggests that damage to CSK structures may contribute to the death of cells treated in G1 but not necessarily to the death of cells treated in S phase.The degree of acrylamide sensitization of heat‐induced CSK disruption was greater for cells exposed to acrylamide prior to spreading than for well‐spread cells. Furthermore, normal spreading of cells was prevented when they were plated into medium containing acrylamide, suggesting that acrylamide interferes with the initial stages of attachment and spreading of these cells. These observations are interpreted in relation to the possible role that VIMFs, together with cortical MFs, may play in mediating cell surface focal contacts in the initial stages of cell attachment and spreading.
Specialized cytoskeletal elements in mammalian eggs: Structural and biochemical evidence for their composition
Authors: McGaughey Robert W. , Capco David G. 
Published: February 2005

AbstractMammalian eggs and embryos contain an extensive detergent‐resistant cytoskeletal network, including many elements which have been referred to as sheets in hamster eggs. In this study we examined the structure of the sheet‐like components by using embedment‐free sections and freeze‐fracture electron microscopy and found that the sheets are composed of both filamentous and particulate components. In addition, exposure to a high salt extraction medium resulted in the disappearance of the sheets at the ultrastructural level. SDS‐polyacrylamide gel electrophoresis of the cell fractions revealed four stainable proteins solubilized by the high salt extraction with one of the proteins being greatly enriched. Because these cytoskeletal sheets undergo an extensive reorganization coincident with key events during early development they serve as internal markers for the establishment of polarity and subsequent differentiation of the first embryonic epithelium, the trophectoderm.
Chemoattractant‐elicited translocation of myosin in motile Dictyostelium
Authors: Nachmias Vivianne T. , Fukui Yoshio , Spudich James A. 
Published: February 2005

AbstractThe distribution of myosin was studied in amebae of the Ax‐3 and NC‐4 strains of Dictyostelium migrating at room temperature, using indirect immunofluorescence of aggregation‐competent amebae and the agar‐overlay technique. Amebae were fixed in methanol‐formaldehyde or absolute acetone at −15°C before or after stimulation with micromolar cyclic AMP at room temperature (20–25°C). Myosin was detected by monoclonal antibodies to Dictyostelium myosin heavy chain followed by a fluorescent secondary antibody that had been preabsorbed to remove nonspecific staining. In both strains there was a striking increase in intensity of anti‐myosin immunofluorescence in the cortex where it appeared as a continuous ring 30 seconds after addition of cyclic AMP. This correlated with a rounding up of the cell body. Sixty seconds after stimulation there was a clear reduction of cytoplasmic myosin rods in conjunction with the increased cortical localization. At this time extensions of largely hyaline cytoplasm were observed that extended beyond the cortical shell of myosin. Two minutes after the stimulus the immunofluorescence remained as a distinct line at the cortex, but the cells began to resume in elongated shape. By 3 minutes (NC‐4 strain) or 5 minutes (Ax‐3 strain) the amebae had largely returned to the control shape, and myosin had returned to its control distribution. Counts of the treated cells at different time points substantiated the observations of individual cells. The time course of translocation of myosin in the Ax‐3 strain parallels the time course of myosin phosphorylation reported in previous studies. The results are interpreted in terms of a working hypothesis for the mechanism of translocation.
Immunolocalization of pericentriolar material in algal mitotic spindles
Authors: La Claire J. W. , Goddard R. H. 
Published: February 2005

AbstractDouble‐label immunofluorescence of tubulin and preicentriolar material (PCM) was carried out with mitotic nuclei in the coenocytic green alga Ernodesmis. Spindle poles are heavily labeled with serum 5051 (anti‐PCM) from midprophase through mid‐ to late anaphase, and bright fluorescence is also evident at the tips of the elongated interzonal spindle in telophase nuclei. Very faint labeling with anti‐PCM is also detected throughout the spindle (and/or its matrix) at all mitotic stages. Control treatments demonstrated that nonspecific surface labeling of chloroplasts with anti‐PCM may be due to some naturally occurring component of human sera rather than to specific labeling by the anti‐PCM serum. Ultrastructural work indicates that the centrosome is always associated with spindle poles through anaphase, but not with the tips of the interzonal telophase Immunoper‐oxidase electron microscopy verifies that anti‐PCM labels the centrosomes of mitotic nuclei in these cells. However, labeling is also present inside the presistent nuclear envelope at the spindle poles, during metaphase, anaphase, and at the tips of the interzonal spindles. Regions of heaviest labeling correspond with amorphous material near the centrioles and at the spindle poles, as evident in conventional electron microscope preparations. The origin of intranuclear amorphous material that labels with anti‐PCM is unclear, but the ends of many spindle microtubules are embedded in it, especially at anaphase, and the tips of microtubules near the amorphous material are often labeled with the antiserum. These results indicate for the first time that serum 5051 does indeed label PCM at the poles of centric spindles in plant cells. Although the location of the labeled material suggests it is associated with the nucleation of spindle microtubules, this conclusion requires more information about microtubule dynamics in these cells. Caution is also warranted in interpreting variant anti‐PCM labeling patterns in other plant cells because of spurious labeling of the spindle itself and other cytoplasmic organelles.
2‐Chloro adenosine triphosphate as substrate for sea urchin axonemal movement
Authors: Omoto Charlotte K. , Brokaw Charles J. 
Published: February 2005

AbstractThe 2‐substituted ATP analog 2‐Chloro ATP was tested for its capacity to support axonemal movement. The movement of sea urchin axonemes reactivated with 2‐CI ATP appeared very similar to that with ATP. Detailed waveform analysis indicated that bend angle and shear amplitude were not significantly different for ATP and 2‐CI ATP. Although wavelength differs at particular nucleotide concentrations, if normalized to the beat frequency, it is similar for ATP and 2‐CI ATP. The main difference in the movement with the two analogs was seen in beat frequency and sliding velocity. The Vmax for beat frequency and mean sliding velocity was lower for 2‐CI ATP. The apparent Km for beat frequency and sliding velocity was much lower for 2‐CI ATP. The ratio of these two effects, that is, (Vmax/Km) is higher for 2‐CI ATP. Thus 2‐CI ATP is a good substrate for axonemal movement. The significantly lower Km of 2‐CI ATP was also demonstrated by its ability to support oscillatory motion at concentrations below that for ATP. The observations identify the structures and conformation of substrate necessary to support axonemal movement.
Indirect immunofluorescence localization of ponticulin in motile cells
Authors: Wuestehube Linda J. , Chia Catherine P. , Luna Elizabeth J. 
Published: February 2005

AbstractPonticulin is the major actin‐binding integral glycoprotein in plasma membranes isolated from log‐phase Dictyostelium discoideum amebae. As such, this protein appears to be an important link between the plasma membrane and actin filaments (Wuestehube and Luna: Journal of Cell Biology 105:1741–1751, 1987). In this study, indirect immunofluorescence microcopy was used to examine the distribution of ponticulin in randomly moving D. discoideum amebae and in amebae engaged in cell migration and phagocytosis. Ponticulin is distributed throughout the plasma membrane and also is present in intracellular vesicles associated with the microtubule‐organizing center‐Golgi complex adjacent to the nucleus. In aggreating amebae, ponticulin is concentrated in regions of lateral cell‐cell contact and in arched regions of the plasma membrane. Ponticulin also is present, but not obviously enriched, in filopodia, in the actin‐rich anterior end of polarized cells, and in detergent‐insoluble cytoskeletons. In amebae engaged in phagocytosis of yeast, ponticulin is present but not enriched in phagocytic cups and is associated with intracellular vesicles around engulfed yeast. These results suggest that ponticulin is stably associated with actin filaments in certain regions of the plasma membrace and that the actin‐binding activity of ponticulin may be tightly controlled.Indirect immupofluorescence microscopy and immunoblot analysis demonstrate that human polymorphonuclear leukocytes also contain a 17 kD protein that specifically cross‐reacts with antibodies affinity‐purified aganst D. discoideum ponticulin. As in D. discoideum, the mammalian 17 kD ponticulin‐analog appears to be localized in plasma membrane and is evident in actin‐rich cell extensions. These results indicate that ponticulin‐mediated linkages between the plasma membrane and actin may be present in higher eukaryotic cells.
Reproductive capacity of sea urchin centrosomes without centrioles
Authors: Sluder G. , Miller F. J. , Rieder C. L. 
Published: February 2005

AbstractFor animal cells, the relative roles of the centrioles and the pericentriolar material (the cenrosomal microtubule organizing center) in controlling the precise doubling of the centrosome before mitosis have not been well defined. To this end we devised an experimental system that allowed us to characterize the capacity of the centrosomal microtubule organizing center to double regularly in the absence of centrioles. Sea urchin eggs were fertilized, stripped of their fertilization envelopes, and fragmented before syngamy. Those activated egg fragments containing just the female pronucleus assembled a monaster at first mitosis. A serial section ultrastructural analysis of such monasters revealed that the radially arrayed microtubules were organized by a hollow fenestrated sphere of electrondense material, of the same appearance as pericentriolar material, that was devoid of centrioles. We followed individual fragments with only a female pronucleus through at least three cell cycles and found that the monasters did not double between mitoses. The observation that fragments with only a male pronucleus repeatedly divided in a normal fashion indicates that the assembly and behavior of monasters were not artifacts of egg fragmentation. Our results demonstrate that the activity that controls the precise doubling of the centrosome before mitosis is distinct and experimentally separable from the centrosomal microtubule organizing center. Our observations also extend the correlation between the reproductive capacity of a centrosome and the number of centrioles it contains (G Sluder and CL Rieder, 1985a: J. Cell Biol. 100:887–896). For a cell that normally has centrioles, we show that a centrosome without centrioles does not reproduce between mitoses.
Preparation of microtubules from rat liver and testis: Cytoplasmic dynein is a major microtubule associated protein
Authors: Collins Christine A. , Vallee Richard B. 
Published: February 2005

AbstractA microtubule associated protein from brain tissue (MAP 1C), has been found to possess many properties in common with ciliary and flagellar dyneins (Paschal et al.: J. Cell Biol. 105:1273–1282, 1987). However, this protein, now designated as cytoplasmic dynein, exhibited several properties which distinguish it from axonemal forms of the enzyme. We have investigated these characteristics further in a study of cytoplasmic dyneins from non‐neuronal tissues. Rat liver and testis in particular were found to contain high levels of cytoplasmic dynein. The yield of dynein from testis was over 70 μg/g of tissue, making this the best source of cytoplasmic dynein of all tissues so far examined. The characterization of dynein from these sources has confirmed and extended our previous observations concerning the unique properties of cytoplasmic dynein. Activation of liver and testis dynein occured at low (<1 mg/ml) tubulin concentration. Polypeptides identified as subunits of brain cytoplasmic dynein (74, 59, 57, 55, and 53 kDa) were present in liver and testis preparations. In addition, polypeptides at 150 and 45 kDa were found to copurify with the non‐neuronal dyneins. The liver and testis enzyme hydrolyzed pyrimidine nucleotides at rates up to 12.5 times faster than ATP, though the relative affinity of cytoplasmic dynein for CTP was much lower (Km = 1.0 mM) than that for ATP. The properties of the testis enzyme were consistent with its identification as a cytoplasmic dynein rather than a sperm axonemal precursor. These data indicate that cytoplasmic dyneins may be widespread in distribution and that they share certain biochemical properties unique from those of axonemal dyneins. These characteristics are consistent with the proposal that cytoplasmic dynein plays a universal role in retrograde organelle motility.