bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dynamic organization of Physarum plasmodium
Authors: Kamiya N. , Allen R. D. , Yoshimoto Y. 
Published: February 2005

AbstractBirefringent fibrils (BRFs) with a positive sign composed of bundles of F‐actin were found throughout the Physarum plasmodium with the mode of existence differing regionally. In the zone behind the leading edge of an advancing plasmodium, where cytoplasmic sol and gel were still not well differentiated, more BRFs came to the foreground when the endoplasm flowed backward (emptying phase), and a substantial portion disappeared when the endoplasm flowed forward (filling phase), except for nodes, from which BRFs were reorganized in the early emptying phase of each cycle. BRFs found in the wall of the streaming channel in the posterior network and the branched vein section ran in parallel to or helically around the channel. They were much more stable and maintained strong birefringence irrespective of the direction of the cytoplasmic flow. When the fan‐like expanse ceased moving forward, the BRFs no longer appeared and disappeared cyclically but persisted in the area which had previously been the front. We concluded that the site of the active contraction‐relaxation rhythm in an advancing plasmodium with antero‐posterior polarity is restricted to its frontal zone and that the rest of the plasmodium is in a state of “tonus” which continuously imparts a certain level of hydrostatic pressure to the interior. The meaning of the tonus and the mechanics of tensile force production in the plasmodium are discussed in terms of a working hypothesis arrived at from the phase relationship between isometric and isotonic contraction waves.
A precursor of the focal contact in cultured fibroblasts
Authors: Izzard Colin S. 
Published: February 2005

AbstractEvidence for a structural precursor of the focal contact in cultured fibroblasts and continuing studies on the development of the precursor and contact are discussed. The structural precursor consists of an F‐actin‐rich, rib‐like fiber within the motile lamellipodium. The focal contact forms beneath the fiber, part of which is retained at the contact as the initial adhesion plaque. Therefore, F‐actin is present at the contact from the beginning. Vinculin accumulates at the plaque during a 90‐second period after the contact forms. A novel feature of the distribution of talin has been found. The protein is present along the distal margin of the lamellipodium, where it is further concentrated as a series of nodes at the tips of each precursor and between precursors. This distribution of talin is independent of that which develops at the plaque after the contact forms. The structural development of the precursor has been followed with AVEC‐DIC optics. The process begins with the development of fine oblique fibers from small structural nodes at the margin of the lamellipodium, and continues with the fusion of the nodes at the margin and inward coalescence of the fibers. It is suggested that talin may function as a cross‐linking protein in the convergence of actin filaments at the membrane, while other actin‐bundling proteins participate in the inward coalescence of the filaments to form fibers. The F‐actin core of the precursor could provide a structural framework against which differences at the external surface of the membrane develop prior to contact formation.
Micromanipulation studies of the mitotic apparatus in sand dollar eggs
Authors: Hiramoto Yukio , Nakano Yoshitaro 
Published: February 2005

AbstractMechanical properties of the mitotic spindle and the effects of various operations of the mitotic apparatus on the chromosome movement and spindle elongation were investigated in fertilized eggs and blastomeres of the sand dollar, Clypeaster japonicus. On the basis of results with mechanical stretching and compression of the spindle with a pair of microneedles and the behavior of an oil drop microinjected into the spindle, it was concluded that the equatorial region of the spindle is mechanically weaker than the half‐spindle region. Anaphase chromosome movement occurred in the spindle from which an aster had been removed or separated with its polar end and in the spindle in which the interzonal region had been removed. This fact indicates that chromosomes move poleward in anaphase by forces generated near the kinetochores in the half‐spindle. Because of the effects of separation or removal of an aster from the spindle on the spindle elongation in anaphase and the behavior of the aster, it was concluded that the spindle elongation in anaphase is caused by pulling forces generated by asters attached to the ends of the spindle.
Dynein as a microtubule translocator in ciliary motility: Current studies of arm structure and activity pattern
Authors: Satir Peter 
Published: February 2005

AbstractThe dynein arms of ciliary doublet microtubules cause adjacent axonemal doublets to slide apart with fixed polarity. This suggests that there is a unique mechanochemistry to the dynein arm with unidirectional force generation in all active arms and also that not all arms are active at once during a ciliary beat. Negative stain and thin‐section images of arms in axonemes treated with β, γ methylene adenosine triphosphate (AMP‐PCP) show a consistent subunit construction where the globular head of the arm interacts with subfiber B of doublet N + 1. This interpretation differs from that provided by freeze etch and STEM interpretations of in situ arm construction and has implications for the mechanochemical cycle of the arm. A computer model of the arms in relation to other axonemal structures has been constructed to test these interpretations. Attachment of the head of the arm to subfiber B is directly demonstrable in splayed axonemes in AMP‐PCP. About half of the doublets in an axoneme show such attachments, while half do not. This might imply that about half the doublets in an axoneme are active at any given instant and can be identified as such. This information may be useful in probing questions of how active arms differ biochemically from inactive arms and of how microtubule translocators in general become active.
Studies on the mechanism of the reversal of rapid organelle transport in myelinated axons of Xenopus laevis
Authors: Smith Richard S. 
Published: February 2005

AbstractRapid 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

AbstractThe 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

AbstractThe 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

AbstractWe 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

AbstractDirected 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

AbstractCaenorhabditis 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

AbstractWe 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

AbstractRecent 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

AbstractIn 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

AbstractCentrosomes 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.