bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the mechanism of the reversal of rapid organelle transport in myelinated axons of Xenopus laevis
Authors: Smith Richard S.
Published: February 2005

Rapid organelle transport was studied by computer‐ and video‐enhanced microscopy in the region of localized lesions in single myelinated axons of Xenopus laevis. Localized lesions were created that were either impermeable to small ions in the bathing medium or were permeable to agents with molecular weights up to 10,000. Providing the axons were bathed in a suitable “internal” medium, organelle transport continued to within a few micrometers of the lesion whether the lesion was permeable or not. Organelles undergoing anterograde and retrograde transport reversed their direction of transport on reaching the lesion. In preparations with lesions that were permeable, nonhydrolyzable analogs of ATP inhibited normally directed and reversed organelle transport. In permeable preparations, vanadate and EDTA inhibited retrograde and reversed retrograde transport at different intra‐axonal concentrations; anterograde and reversed anterograde transport were also differentially inhibited. Anterograde and retrograde organelle transport were also shown to be inhibited at different intraaxonal concentrations of vanadate and EDTA.

The results provide evidence for the existence of two different axonal transport mechanisms in myelinated axons. The two mechanisms can account for the normally directed and reversed transport of individual organelles.
Rapid axonal transport as a chromatographic process: The use of immunocytochemistry of ligated nerves to investigate the biochemistry of anterogradely versus retrogradely transported organelles
Authors: Dahlströ Annica B. , Bööj Serney
Published: February 2005

The distribution and axonal transport of cholinergic organelles has been studied in the rat motor system, using immunofluorescence methods and a cytofluorimetric technique for quantification of immunoreactive material. Crush‐operated spinal roots and sympathectomized sciatic nerves were sectioned longitudinally and incubated with antisera against p38, SV2, CGRP, chromogranin A (Chr A), synapsin I (SYN I), and with RASVA (rabbit anti‐synaptic vesicle antiserum). Motor endplates were also studied. It was observed that proximally accumulating organelles—i.e., organelles which were in transport distally in the axons— contained RASVA‐like immunoreactivity (LI) p38, SV2, CGRP‐LI, Chr A‐LI, and SYN I‐LI. Retrogradely transported organelles, however, contained only p38 and SV2 in addition to RASVA‐LI, but virtually no CGRP‐LI, ChrA‐LI, or SYN I‐LI. It is suggested that the rapid axonal transport mechanism operates in the nerves like a chromatographic process, which allows the concentration in the axons, proximal or distal to the crush, of organelles in anterograde or retrograde transport, respectively. The technique of nerve crushes in combination with immunocytochemistry can therefore be used to investigate the biochemical composition of organelles in transit along the axon, and give information on neurobiological events occurring in these long processes leading to the nerve endings. In this study, biochemical differences between anterogradely and retrogradely transported cholinergic crganelles in the motor neuron of the rat have been observed, and were related to suggested events in the endplate.
Flagellar quiescence and transience of inactivation induced by rapid pH drop
Authors: Sato Fumi , Mogami Yoshihiro , Baba Shoji A.
Published: February 2005

The effects of rapid pH drop on the flagellar movement of reactivated sea urchin sperm were studied by video microscopy and by a newly developed pH jump method. Triton‐demembranated sperm were reactivated in a thin layer of the reactivation medium containing ATP and potassium acetate and supported by a ring‐shaped Millipore filter stuck to the lower surface of a supported coverslip. The pH of the medium was lowered rapidly by dissolving acetic acid vapor abruptly introduced into a gap between the cover and slide. Flagellar beating ceased immediately when the pH of the reactivation medium was lowered. At least two types of cessation were distinguished: (1) “instantaneous” cessation in a bent form closely resembling those characteristic of steady‐state beating before pH drop (waveform freeze), and (2) flagellar quiescence in a cane‐shaped form resembling those characteristic of Ca‐induced quiescence (cane‐shaped quiescence). The flagellum again began beating if the pH was raised to normal but eventually was disintegrated by tubule sliding if the pH was left lowered. Field‐by‐field analysis of the transient movement of flagella becoming quiescent upon pH drop demonstrated that the proximal bend of the cane‐shaped form corresponded to the principal bend of the steady‐state beating in some flagella, but in others, to the reverse bend. These observations indicate that low pHs affect flagellar beating by interfering with sliding‐bending conversion by a mechanism different from that previously reported.
Characteristic structures of actin gels induced with hepatic actinogelin or with chicken gizzard alpha‐actinin: Implication for their function
Authors: Tsukita Sachiko , Mimura Naotoshi , Tsukita Shoichiro , Khono Kenichi , Ohtaki Tetsuya , Oshima Teruyo , Ishikawa Harunori , Asano Akira
Published: February 2005

We studied the properties of actinogelin, a Ca2+‐regulated actin cross‐linking protein isolated from Ehrlich tumor cells or rat liver. Chicken gizzard α‐actinin was used as a Ca2+‐insensitive control. Actinogelin, which has very high gelation activity under low Ca2+ conditions, was found using electron microscopic or fluorescence studies to induce formation of a characteristic structure in which actin filaments and bundles radiate to (or converge from) all directions from spot‐like core structures. A similar structure was induced with actinogelin, even in the presence of 0 7 saturation of tropomyosin. No such structure was detected with actinogelin under high Ca2+ conditions, and only a few were found with gizzard α‐actinin. Because reconstituted structures are similar to those observed intracellularly, actinogelin may be important in the formation of similar microfilament organization in the cells. It seems also important that these structures are reconstituted with only two purified protein components, i.e., actinogelin and actin.

Immunocompetition studies showed that actinogelin and gizzard α‐actinin partially shared antigenicity, and their molecular shape and peptide maps were similar. Their amino acid compositions [Kuo et al., 1982], subunit and domain structures, and binding sites on actin [Mimura and Asano, 1987] are also very similar. Therefore, it is concluded that actinogelin belongs to α‐actinin superfamily proteins. Furthermore, the presence of functionally different subfamilies concerned with Ca2+ sensitivy, gelation‐efficiency, and others is discussed. Actinogelin, which induces networks of actin filaments, may be classified as high gelation type.
Cultured cell extracts support organelle movement on microtubules in vitro
Authors: Dabora Sandra L. , Sheetz Michael P.
Published: February 2005

Directed movements of organelles have been observed in a variety of cultured cells. To study the regulation and molecular basis of intracellular organelle motility, we have prepared extracts from cultured chick embryo fibroblasts (CEF cells) which support the movement of membranous organelles along microtubules. The velocity, frequency and characteristics of organelle movements in vitro were similar to those within intact cells. Organelles and extract‐coated anionic beads moved predominantly (80%) toward the minus ends of microtubules that had been regrown from centrosomes, corresponding to retrograde translocation. Similar microtubule‐dependent organelle movements were observed in extracts prepared from other cultured cells (African green monkey kidney and 3T3 cells).

Organelle motility was ATP and microtubule dependent. The frequency of organelle movement was inhibited by acidic (pH<7) or alkaline (pH>8) solutions, high ionic strength ([KCl] = 0.1 M), and the chelation of free magnesium ions. Treatment of the extracts with adenylyl imidodiphosphate (AMP‐PNP, 7 mM), sodium orthovanadate (vanadate; Na3VO4, 20 μM), or N‐ethylmaleimide (NEM, 2 mM) blocked all organelle motility. The decoration of microtubules with organelles was observed in the presence of AMP‐PNP or vanadate. Motility was not affected by cytochalasin D (2 μM) or cAMP (1 mM). Kinesin (Mr= 116,000), an anterograde microtubule‐based motor, was partially purified from the CEF extract by microtubule affinity purification in the presence of AMP‐PNP, and was able to drive the movement of microtubule on glass coverslips. A similar preparation made in the presence of vanadate contained a different subset of proteins and did not support motility. These results demonstrate that intracellular organelle motility can be reproduced in vitro and provide the basis for investigating the roles of individual molecular components involved in the organelle motor complex.
Relationship between plasma membrane mobility and substrate attachment in the crawling movement of spermatozoa from Caenorhabditis elegans
Authors: Pavalko Fredrick M. , Roberts Thomas M. , Holliday L. Shannon
Published: February 2005

Caenorhabditis elegans sperm are nonflagellated cells that lack actin and myosin yet can form pseudopods to propel themselves over solid substrates. Surface‐attached probes such as latex beads, lectins, and antimembrane protein monoclonal antibodies move rearward over the dorsal pseudopod surface of sessile cells. Using monoclonal antibodies against membrane proteins of C. elegans sperm to examine the role of localized membrane assembly and rearward flow in crawling movement, we determined that substrates prepared by coating glass with antimembrane protein antibodies, but not naked glass or other nonmembrane‐binding proteins, promote sperm motility. Sperm locomotion is inhibited in a concentration‐dependent fashion when cells are bathed with soluble antimembrane protein monoclonal antibodies but not with antimouse Ig antibodies or a monoclonal antibody against a sperm cytoplasmic protein. Our results suggest that C. elegans sperm crawl by gaining traction with substrate‐attached ligands via their surface proteins and by using the motor that moves those proteins rearward on unattached cells to pull the entire cell forward. Continuous insertion of new proteins at the front of the cell and their subsequent adhesion to the substrate allows this process to continue.
Micromanipulation studies of the asyrnmetric positioning of the maturation spindle in Chaetopterus sp. Oocytes: I. Anchorage of the spindle to the cortex and migration of a displaced spindle
Authors: Lutz Douglas A. , Hamaguchi Yukihisa , Inoué Shinya
Published: February 2005

We investigated the nature of the asymmetric positioning and attachment of Chaetopterus oocyte meiotic spindles to the animal pole cortex by micromanipulation. The manipulated spindle's behavior was analyzed in clarified oocyte fragments using video‐enhanced polarized light microscopy. As the spindle was drawn towards the cell interior with a microneedle, the cell surface dimpled inwards adjacent to the outer spindle pole. As the spindle was pulled further inwards, the dimple suddenly receded indicating a rupture of a mechanical link between the cell cortex and outer spindle pole. The spindle paused briefly when released from the microneedle; then it spontaneously migrated back to the original attachment site and reassociated with the cell cortex. Positive birefringent astral fibers were seen running between the outer spindle pole and the cortex during the migration. The velocity of the spindle during its migration tended to increase as it came closer to the cortex. Velocities as high as 1.25 μm/sec. were measured. If removed too far from the attachment site cortex ( > 35 μm), the spindle remained stationary until pushed closer to the original attachment site. Spindles, inverted by micromanipulation, migrated and reattached to the cortical site by their former inner pole; thus either spindle pole can seek out and migrate to the original attachment site. However, spindle poles pushed against other cortical regions did not attach demonstrating that there is only one unique, localized attachment site for spindle attachment.
Spindle microtubule dynamics: Modulation by metabolic inhibitors
Authors: Wadsworth P. , Salmon E. D.
Published: February 2005

Recent experiments have shown that spindle microtubules are exceedingly dynamic. Measurements of fluorescence recovery after photobleaching (FRAP), in cells previously microinjected with fluorescent tubulin, provide quantitative information concerning the rate of turnover, or exchange, of tubulin subunits with the population of microtubules in living cells at steady state. In an effort to elucidate the pathways and factors that regulate tubulin exchange with microtubules in living cells, we have investigated the energy requirements for tubulin turnover as measured by FRAP. Spindle morphology was not detectably altered in cells incubated with 5 mM sodium azide and 1 mM 2‐deoxyglucose (Az/DOG) for 5 minutes, as assayed by polarized light microscopy and antitubulin immunofluorescence. In FRAP experiments on these ATP‐depleted cells, the average rate of recovery and the average percent of bleached fluorescence recovered were reduced to 37% and 30% of controls, respectively. When the inhibitors were removed, cells continued through mitosis, and rapid FRAP was restored. In the presence of azide and glucose, the rate of recovery and percent of fluorescence recovered were only slightly reduced, demonstrating that energy production via glycolysis can support microtubule turnover. Longer incubations with Az/DOG altered the microtubule organization in mitotic cells: astral microtubules lengthened and spindle fibers shortened. Furthermore, both astral and spindle microtubules became resistant to nocodazole‐induced disassembly under these conditions. Together these observations indicate that microtubule dynamics require ATP and suggest a relationship between microtubule organization and turnover.
Cytoskeletal architecture and immunocytochemical localization of fodrin in the terminal web of the ciliated epithelial cell
Authors: Kobayashi Naoto , Hirokawa Nobutaka
Published: February 2005

In order to understand the cytoskeletal architecture at the terminal web of the ciliated cell, we examined chicken tracheal epithelium by quick‐freeze deep‐etch (QFDE) electron microscopy combined with immunocytochemistry of fodrin. At the terminal web, the cilia ended into the basal bodies and then to the rootlets. The rootlets were composed of several filaments and globular structures attached regularly to them. Decoration with myosin subfragment 1 (S1) revealed that some actin filaments ran parallel to the apical plasma membrane between the basal bodies, and other population traveled perpendicularly or obliquely, i.e., along the rootlets. Some actin filaments were connected to the surface of the basal bodies and the basal feet. Among the basal bodies and the rootlets there existed three kinds of fine crossbridges, which were not decorated with S1. In the deeper part of the terminal web, intermediate filaments were observed between the rootlets and were sometimes crosslinked with the rootlets. Immunocytochemicstry combined with the QFDE method revealed that fodrin was a component of fine crossbridges associated with the basal bodies. We concluded that an extensive crosslinker system among the basal bodies and the rootlets along with networks of actin and intermediate filaments formed a structural basis for the effective beating of cilia.
Microtubules are required for centrosome expansion and positioning while microfilaments are required for centrosome separation in sea urchin eggs during fertilization and mitosis
Authors: Schatten Heide , Walter Marika , Biessmann Harald , Schatten Gerald
Published: February 2005

Centrosomes undergo cell cycle‐dependent changes in shape and separations, changes that govern the organization of the cytoskeleton. The cytoskeleton is largely organized by the centrosome; however, this investigation explores the importance of cytoskeletal elements in directing centrosome shape. Since the sea urchin egg during fertilization and mitosis displays dramatic and synchronous changes in centrosome shape, the effects of cytoskeletal inhibitors on centrosome compaction, expansion, and separation were explored by the use of anticentrosome immunofluorescence microscopy. Centrosome expansion and separation was studied during two phases: the transition after sperm incorporation, when the compact sperm centrosome enlarges and the sperm aster develops, and from prometaphase to telophase, when the compact spindle poles enlarge. Compaction was investigated when the dispersed centrosome at interphase condenses into the two spindle poles at prometaphase. Although centrosome expansion and separation typically occur concurrently, β‐mercaptoethanol results in centrosome separation independent of expansion. Microtubule inhibitors prevent centrosome expansion and separation, and expanded centrosomes collapse. Since pronuclear union is arrested by microtubule inhibitors, this treatment also affords the opportunity to explore the relative attractiveness of the male and female pronuclei for these centrosomal antigens. Both pronuclei acquire centrosomal material; though only the male centrosome is capable of organizing a functional bipolar mitotic apparatus at first division, the female centrosome nucleates a monaster. Microfilament inhibition (cytochalasin D) prevents centrosome separation but not expansion or compaction. These results demonstrate that as the centrosome shapes the cytoskeleton, the cytoskeleton alters centrosome shape.
Polymerization of additional actin is not required for capping of surface antigens in B‐lymphocytes
Authors: Jackman W. T. , Burridge K.
Published: February 2005

CH12 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

Macrocilia 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

The 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

The 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

A 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

The 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

Mammalian 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

The 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

Double‐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

The 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

Ponticulin 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

For 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

A 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.
Kinetics of granulocyte phagocytosis: Rate limited by cytoplasmic viscosity and constrained by cell size
Authors: Evans Evan
Published: February 2005

Micromanipulation of yeast particles and blood granulocytes has been used to study the kinetics of single phagocytosis events. The ingestion process was quantitated by observation of sequential adhesion and encapsulation times. Both adherence and encapsulation times were found to increase greatly as the temperature was reduced below 37°C calcium in solution facilitated adhesion of the particle to the phagocyte but not encapsulation; both adhesion and encapsulation processes required a minimum level of plasma components (presumably complement). The general nature of these observations were confirmatiory of previous studies, but this study is unique in that the specific time course of single particle ingestion was quantitated. It was immediately apparent that the phagocytosis process was 100% efficient above the threshold concentrations required for plasma and temperature, but variations in times from cell to cell indicated heterogeneity in the population. The total time for ingestion varied from as low as 2 sec/particle at 37°C to above several min/particle below 15°C. Encapsulation times for particles were normalized by estimates of particle surface areas to establish a specific time/unit area of particle surface: from 0.5 sec/10−8 cm2 at 37°C to greater than 8 sec/10−8 cm2 at 15°C. The temperature dependence of the encapsulation time correlated well with the temperature dependence of the “apparent” viscosity for granulocytes measured by micropipet aspiration. As such, the kinetic properties observed in these phagocytosis tests are consistent with a model that both assembly of the contractile system and the displacement of the surface by active contraction in phagocytosis are limited by viscous dissipation in the cell. Based on temperature dependence of the adhesion time, the activation energy associated with “turning on” the contractile system is quite large, with a value of about 31 kcal/mole. Finally, it was found that serial “feeding” of yeast to a single granulocyte satiated the phagocyte only after six to eleven particles had been encapsulated. The number limit to ingestion was directly proportional to inital size of the granulocyte.
The cytoskeleton of the naked green flagellate Spermatozopsis similis: Isolation, whole mount elecron microscopy, and preliminary biochemical and immunological characterization
Authors: Lechtreck K.‐F. , McFadden G. I. , Melkonian M.
Published: February 2005

The cytoskeleton of the naked, biflagellate green alga Spermatozopsis similis Preisig & Melkonian was isolated by treatment of cells with Nonidet P‐40 (0.1%) in lysis buffer (30 mM HEPES, 5 mM EGTA, 15 mM KCl, pH 7) and studied in detail by whole‐mount electron microscopy. Isolated cytoskeletons retain the twisted shape of live cells and consist of the two axonemes, the basal apparatus with 4 microtubular and two fibrous roots, and 8–10 secondary cytoskeletal microtubules (SCMT's). The four microtubular flagellar roots differ in number of microtubules (two types with 2 or 5 microtubules, respectively), in their association with fibrous roots of the system I‐type (two‐stranded roots), in total length (two roots with an average of 4.5 μm and two roots with and average of 7.5 μm), and in length of individual root microtubules. Certain of the root microtubules and most of the SCMT's extend to the posterior end of the cell where they converge, terminate and are interconnected by a fibrous cap‐like structure, the rhizosyndesmos. This novel structure consists of a network of 2 nm filaments that presumably lacks centrin as indicated by double immunofluorescence (anti‐α‐tubulin and anti‐centrin) of isolated cytoskeletons. Two‐dimensional gel electrophoresis of isolated, purified basal apparatuses of S. similis identifies among other proteins two isoforms of centrin and α‐ and β‐tubulin as intrinsic components.
Formation of miniasters in the cytoplasm of hexyleneglycol‐treated sea urchin eggs
Authors: Endo Sachiko , Toriyama Masaru , Ohta Kunihiro , Sakai Hikoichi
Published: February 2005

Miniasters formed in mitotic sea urchin egg after treatment with 5% hexylene‐glycol were investigated with the combined techniques of indirect immunofluo‐rescence using anti‐tubulin and anti‐51 kD protein antibodies and electron microscopy.

The formation of miniasters was dependent on the mitotic cycle. In the cytoplasm of eggs treated with hexyleneglycol at early prometaphase, a small number of microtubule fragments was observed, whereas in those treated at pro‐metaphase, many miniasters and microtubule fragments were seen. When treated at metaphase, we found a great number of miniasters: 250–350 in one egg. In contrast, no miniasters were seen in eggs treated at anaphase, although many long microtubules that spread throughout the cytoplasm were observed. In the eggs treated at telophase, we scarcely noticed microtubule structures in the cytoplasm. In the center of miniasters, granules were found, showing the same size and electron density as those of the microtubule‐organizing granules (MTOGs). Furthermore, the 51 kD protein, a component of the centrosome and mitotic spindle, was observed to be localized in the region of miniasters.
Identification and immunolocalization of cytoplasmic dynein in dictyostelium
Authors: Koonce Michael P. , Richard McIntosh J.
Published: February 2005

A high molecular weight microtubule binding protein has been isolated from homogenates of Dictyostelium. Because of its sedimentation velocity (20s), ATP‐sensitive binding to microtubules. UV‐vanadate‐ATP mediated fragmentation, prominent CTPase activity, and its ability to produce limited microtubule movement in vitro, we consider this protein to be a form of cytoplasmic dynein. A polyclonal antibody monospecific to this protein was produced, and dynein's intracellular distribution in ameboid cells was examined by immunofluorescence. The antibody labels a punctate cytoplasmic pattern, localizes to a spherical region adjacent to the nucleus, and also appears to label the nuclei. The punctate staining pattern is consistent with cytoplasmic dynein's proposed function in organelle transport. The spherical juxtanuclear object stained is coincident with this cell's microtubule organizing center, an obvious termination point for minus‐end directed microtubule motors. By immunofluorescence, there does not appear to be a substantial amount of dynein in the intranuclear mitotic spindles of Dictyostelium, These data provide evidence for localization of cytoplasmic dynein in cells, and suggest that Dictyostelium will be a useful system in which to study the molecular biology of microtubule‐associated motor enzymes.