bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro induction of crawling in the amoeboid sperm of the nematode parasite, Ascaris suum
Authors: Sepsenwol Sol , Taft Stephen J.
Published: February 2005

In a highly synchronous process, the immotile spermatids of Ascaris suum extend pseudopods and become rapidly crawling sperm when treated with an extract from the glandular vas deferens of the male under strict anaerobic conditions. Within 9–12. min, a pseudopod develops, elongates rapidly, and exhibits a continuous flow of membrane specializations, the villipodia, from tip toward base. When attached to acid‐washed glass, the pseudopod pulls the cell body along at speeds exceeding 70 μm/min. The pseudopod length remains constant while retrograde flow of villipodia proceeds at the same rate as the sperm's forward movement. Cohorts of about 15 villipodia form at the leading edge, move reaward together, and disappear at the junction of pseudopod and cell body. These are the termination of branched, refringent fibers, which extend the length of the pseudopod. The latter are the fiber complexes that form its cytoskeleton (Sepsenwol et al.: Journal of Cell Biology 108:55–66, 1989). Locomoting cells sometimes change direction when another crawls by and follow each other. When cells are exposed to air, forward movement ceases in a predictable pattern: the forward extension of the leading edge ceases, the pseudopod shortens from the base, and the cell body continues to be pulled forward. These data contribute to a model for Ascaris sperm amoeboid motility in which independent processes of continuous extension at the leading edge and continuous shortening at the base of the pseudopod act to propel the cell forward.
Keratin filaments restrict organelle migration into the forming spindle of newt pneumocytes
Authors: Mandeville Elizabeth C. , Rieder Conly L.
Published: February 2005

When viewed by light microscopy the mitotic spindle of newt pneumocytes appears to assemble in an optically clear area of cytoplasm, virtually devoid of mitochondria and other organelles, which is often much larger than the spindle. This clear area is also frequently larger than the region previously occupied by the nucleus. It forms even in prometaphase cells depleted of microtubules prior to nuclear envelope breakdown by colchicine treatment. Time‐lapse video microscopy reveals that as prometaphase proceeds this clear area slowly and progressively collapses around the forming spindle so that it is greatly diminished or nonexistent by the onset of anaphase. The sharply defined nature of the boundary between the clear area and the remaining cytoplasm and the fact that organelles accumulate at its periphery suggest that a structural barrier is present at the boundary that limits organelle migration into the forming spindle. Immunofluo‐ rescence and electron microscopy, of cells previously followed in the living state, reveal that the periphery of the clear area contains prominent bundles of keratin filaments but lacks microtubules and actin. From our observations we conclude that keratin filaments form a loosely organized cage that surrounds the forming newt pneumocyte spindle. We propose that this cage functions, in part, to restrict the dispersion of chromosomes during nuclear envelope breakdown and to impede the bulk migration of organelles into the forming spindle.
The outer arm dynein α‐heavy chains of sea urchin sperm flagella and embryonic cilia are different
Authors: Ogawa Kazuo , Yokota Etsuo , Hamada Yoshio , Wada Shigeo , Okuno Makoto , Nakajima Yoko
Published: February 2005

Sea urchin sperm outer arm dynein is a multi‐subunit protein composed of heavy chains, intermediate chains, and light chains. We prepared monoclonal and affinity‐purified polyclonal antibodies to heavy and intermediate chain subunits and examined whether the embryonic ciliary axonemes ofthe same species contain the polypeptides sharing epitopes with them. Ciliary axonemes contained a high molecular weight polypeptide with the exact same mobility as flagellar β‐heavy chain. This polypeptide also shared epitopes with it. In contrast, no polypeptide had the exact same molecular weight as flagellar α‐heavy chain and shared epitopes with it. Western blots showed that ciliary axonemes also contain three polypeptides sharing epitopes with the respective flagellar intermediate chain. The present results revealed that the α‐heavy chains of flagellar and ciliary outer arm dyneins are different.
Nuclear and mitotically enhanced epitope
Authors: Chriss Hoffman John , Michael Mullins J.
Published: February 2005

Salt‐extracted proteins of taxol‐stabilized microtubules from Chinese hamster ovary cells arrested at mitosis were used to immunize mice for hybridoma production. From a group of related monoclonal antibodies (MAbs), one, C9, recognized an epitope on antigens localized by immunofluorescence microscopy to interphase centrosomes and nuclei. The availability of the nuclear antigen was cell cycle‐dependent; however, permeabilization of cells before fixation revealed that the antigen was present throughout the cell cycle. The nuclear antigen was exposed during prophase and was released from the nucleus upon nuclear envelope breakdown filling the cytoplasm of the mitotic cell. Antigenic material re‐accumulated at daughter nuclei and was concealed during Gl phase. Detergent extraction of the cytoplasmic antigen from mitotic cells enabled localization of antigens to centrosomes, kinetochores, and the furrowing region/midbody. Immunoblot analysis of cells of a variety of species of origin identified an approximate 250 kD polypeptide as corresponding to the nuclear antigen, whereas polypeptides of 107/117 kD as well as approximately 250 kD accounted for the mitotic cytoplasmic antigens. No polypeptides could be associated with antigens at centrosomes, kinetochores, or midbodies. This MAb joins the antibody preparations previously reported that describe nuclear antigens, or epitopes on antigens, enhanced at mitosis.
T‐1, a mitotic arrester, alters centrosome configurations in fertilized sea urchin eggs
Authors: Itoh Tomohiko J. , Schatten Heide , Schatten Gerald , Mazia Daniel , Kobayashi Akio , Sato Hidemi
Published: February 2005

T‐1 induces modifications in the shape of the centrosome at division in fertilized eggs of the North American sea urchin, Lytechinus pictus. Phase contrast microscopy observations of mitotic apparatus isolated from T‐1treated (1.7–8.5μM) eggs at first division shows that the centrosomes already begin to spread or to separate by prophase and that the mitotic spindle is barrel‐shaped. When eggs are fertilized with sperm that have been pretreated with T‐1, the centrosomes become flattened; the spindles are of normal length. Immunofluorescence microscopy using an anti‐centrosomal monoclonal antibody reveals that T‐l modifies the structure of the centrosome so that barrel‐shaped spindles with broad centrosomes are observed at metaphase, rather than the expected focused poles and fusiform spindle. Higher concentrations of T‐l induce fragmentation of centrosomes, causing abnormal accumulation of microtubules in polar regions. These results indicate that T‐l directly alters centrosomal configuration from a compact structure to a flattened or a spread structure. T‐l can be classified as a new category of mitotic drugs that may prove valuable in dissecting the molecular nature of centrosomes.
Propranolol, a β‐adrenergic receptor blocker, affects microfilament organization, but not microtubules, during the first division in sea urchin eggs
Authors: Nicotra Antonietta , Schatten Gerald
Published: February 2005

Propranolol, a β‐adrenergic receptor blocker, blocks the formation of the cleavage furrow, while karyokinesis is unaffected during first division in the sea urchins Paracentrotus lividus or Lytcchinus pictus. This effect is reversed by adrenalin, indicating that it is mediated by an adrenergic mechanism. The staining of F‐actin microfilaments by rhodamine phalloidin in eggs in which the cleavage is blocked by the drug has revealed that propranolol affects both the distribution and the organization of actin microfilaments. A low‐voltage scanning electron microscopy (LVSEM) study of microvilli in these eggs shows an extensive rearrangement of the egg surface. Anti‐tubulin immunofluorescence microscopy of eggs treated with propranolol shows that they form normal mitotic asters. This indicates that while cleavage is affected, mitotic spindle formation is not. These results suggest that neurotransmitter monoamines known to be present in the sea urchin egg might be involved in the reorganization of the actin cytoskeleton underlying the formation of the cleavage furrow.
1,2‐dioctanoylglycerol accelerates or retards mitotlc progression in Tradescantia stamen hair cells as a function of the time of its addition
Authors: Larsen Paul M. , Wolniak Stephen M.
Published: February 2005

We have treated living, intact stamen hair cells from the spiderwort plant, Tra‐descantia virginiana, with 0.5 μg/ml or 60 μg/ml 1,2‐dioctanoylglycerol, a potent and permeant activator of protein kinase C, and have observed the rates of progression of mitosis from prophase through anaphase. We have found that in addition to the concentration used, the time of initial treatment with 1,2‐di‐octanoylglycerol defines the response by the cells. The cells rapidly undergo nuclear envelope breakdown when this diglyceride is added in very late prophase, 0 to ∼8 min prior to the time of normal nuclear envelope breakdown. Anaphase onset occurs 28 min after nuclear envelope breakdown, rather than after the 33 min interval observed in untreated cells. Rapid progression through metaphase is also observed if cells are treated with 0.5 μg/ml 1,2‐dioctanoylglycerol during prometaphase, up to 15 min after nuclear envelope breakdown. The addition of 0.5 μg/ml 1,2‐dioctan oylglycerol in late metaphase, ∼26 min after nuclear envelope breakdown, results in sister chromatid separation slightly ahead of its normal time, 33 min after nuclear envelope breakdown, and in precocious cell plate vesicle aggregation, 3–5 min earlier than that observed in untreated cells. Treatment of cells with 60 μg/ml of 1,2‐dioctanoylglycerol at any point during the interval from 0 to ∼5 min prior to nuclear envelope breakdown results in precocious entry into anaphase. If cells are treated with either 0.5 μg/ml or 60 μg/ml 1,2‐dioctanoylglycerol earlier than 20 min before nuclear envelope breakdown, they do not enter mitosis, but instead revert to interphase without dividing. When 1,2‐dioctanoylglycerol is added atother times during mitosis, the rate of subsequent mitotic progression is dramatically slowed; the cells require >55 min to progress from nuclear envelope breakdown to anaphase onset, though once in anaphase, the cells progress onward to cytokinesis at normal rates. Treatments of cells with 1,3‐dioctanoylglycerol at any point during prophase, prometaphase, or metaphase are without effect on the rate of subsequent mitotic progression. The shifts in response by cells treated at specific times with 1,2‐dioctanoylglycerol during mid‐ and late metaphase may be indicative of the existence of one or more regulatory switch points (i.e., checkpoints) just prior to anaphase onset.
Increasing tubC β‐tubulin synthesis by placing it under the control of a benA β‐Tubulin upstream sequence causes a reduction in benA β‐tubulin level but has no effect on microtubule function
Authors: May Gregory S. , Waring Richard B. , Morris N. Ronald
Published: February 2005

We have constructed a chimeric β‐tubulin gene that places the structural gene for the tubC β‐tubulin of Axpergillus nidulans under the control of the benA β‐tubulin promoter. Introduction of cither this chimeric gene or a second wild‐type ben.A gene into a benomyl‐resistant benA22 strain causes it to become benomyl sensitive, indicating that the introduced genes are functional. Analysis of the tubulin proteins synthesized in benA22 strains into which a second wild‐type benA β‐tubulin gene was transformed showed that the total amount of β‐tubulin protein was the same as in the parental strain with a single benA gene. Thus the level of β‐tubulin must be regulated. This was also true of transformants carrying an extra copy of the chimeric β‐tubulin gene. The total amount of β‐tubulin was the same as in the parental strain. Two‐dimensional gel analysis showed that the endoge‐nous benA22 and the introduced chimeric tubC gene contributed equally to the total β‐tubulin pool. Th; fact that one‐half of the benA β‐tubulin could be replaced by tubC β‐tubulin with no effect on the growth of the cells suggests that the benA and tubC β‐tubulins are functionally interchangeable.
Heterogeneity of microtubules in dividing sea urchin eggs revealed by immunofluorescence microscopy: Spindle microtubules are composed of tubulin isotypes different from those of astral microtubules
Authors: Oka Mikako T. , Arai Takao , Hamaguchi Yukihisa
Published: February 2005

The heterogeneity of mitotic microtubules in dividing sea urchin eggs was investigated by indirect immunofluorescence using five anti‐α‐tubulin (YL1/2, DM1A, E3B8, D2D6, and 6‐11B‐1) and two anti‐β‐tubulin (E6B6 and DM1B) antibodies. These antibodies were divided into four classes in regard to the different immunofluorescent staining patterns: class I, which strongly stained both the spindle and aster (YL1/2, DM1A, E3B8 and E6B6); class II, which strongly stained the spindle but weakly stained the aster (D2D6); class III, which stained only the aster (DM1B); and class IV, which did not stain the mitotic apparatus (6‐11B‐1). These results suggest that tubulin isotypes are distributed differently in the sea urchin mitotic microtubules and that α‐tubulin isotype(s) recognized by D2D6 is (are) localized mainly in spindle microtubules, whereas β‐tubulin isotype(s) recognized by DM1B is (are) found only in astral microtubules.
Depolarization‐controlled parameters of the ciliary cycle and axonemal function
Authors: Sugino Kazuyuki , Machemer Hans
Published: February 2005

Depolarization‐induced cycles of a frontal cirrus of Stylonychia were investigated by applying methods of axial‐view analysis of the cilia, high‐speed microcinématography, and step voltage‐clamp. Rising depolarization (from 3 mV to 7ge; 30mV) increased the rate of beating from zero to maximally 58 Hz. During cyclic activity, the axis of the beat cone of a proximal segment of the cirrus was inclined by 60° (0° = perpendicular to cell surface), and was always oriented 90° counterclockwise to the power stroke. With the stimulus amplitude rising, the orientations of the power stroke and inclination were increasingly shifted in more counterclockwise directions by up to 80° After correction for inclination ( = normalization), and following planification of the track of the segment, we determined the following properties of the cycle during depolarization: The course of the cycle tended to be rounded, i.e., the ratio of major over minor amplitudes (= spatial polarity) approximated a value of 1.6 which is only two thirds of maximal spatial polarity observed during hyperpolarization. The angular velocity generally increased with rising steps of depolarization; up to +5 mV (and comparable to hyperpolarization‐induced responses), the velocity maximum occurred during the return stroke. With depolarizations ≥7 mV the angular velocity maximum shifted to the power stroke so that the temporal polarity (rates of power stroke over rates of return stroke) increased from 0.4 to 1.6. Calculations of the angular velocity as referred to the proximal ciliary segment level suggest active sliding rates (between 5 and 30 nm/ms) of identified pairs of doublet microtubules. Ciliary frequency is a function of the rate of reorientation of the cyclic track; this parameter, which corresponds to the rate of translocation of active sliding between pairs of doublets, grew with the amplitude of depolarization. Translocation rates were high during transitions between the beat phases (power stroke, return stroke), and were reduced during these phases. Orientational polarograms of the mean rates of both active sliding and sliding translocation show properties of discreteness as well as continuity. The depolarization‐induced changes in inclination, and the inferred patterns of sliding rate and sliding translocation rate, are compared with previous results from hyperpolarization‐dependent activation of the same motor organelle.
Effects of trypsin and low Ca2+ on zonulae adhaerentes between chick retinal pigment epithelial cells in organ culture
Authors: Sandig Martin , Hergott Greg J. , Kalnins Vitauts I.
Published: February 2005

The junctional complexes in chick retinal pigment epithelial (RPE) cells in situ contain unusually large zonulae adhaerentes (ZAs) composed of subunits termed zonula adhaerens complexes (ZACs). To determine whether the properties of the ZAs differ between RPE cells which contain ZACs, and MDCK cells which lack ZACs, we investi‐gated the effects of treatment with trypsin and/or low Ca2+ by transmission electron microscopy and staining for F‐actin. Treatment of RPE cells for 1 h with trypsin alone has no apparent effect on the morphology of the ZA in either MDCK or RPE cells. In contrast to the ZAs in MDCK cells, which split after 3 min in low Ca2+, the ZAs in chick RPE cells stay intact even after 2 h, although the intermembrane discs, i.e., the extracellular components of the ZACs, are no longer visible. After 30 min of treatment with trypsin and low Ca2+, the ZAs split in both cell types. The CMBs start to contract, translocate toward the cell interior, and eventually disappear. This process continues even when the RPE cells are returned to normal medium. New ZAs, composed of ZACs, form between RPE cells 3 h after return to normal medium. These findings suggest that the ZACs in the ZAs of RPE cells are not directly responsible for the increase in resistance to low Ca2+. They also show that the ZA‐junctions in RPE cells are not only structurally different from those previously examined, but also behave differently in response to experimental manipulation.
Behavior of C‐shaped microtubule endings in the cell
Authors: Wolf Klaus Werner
Published: February 2005

The association of incomplete microtubule assemblies with either another incom‐plete structure or complete microtubules was studied in two organisms, the phytoflag‐ellate Polytoma papillatum and the phorid fly Megaselia scalaris, using transmission electron microscopy. In the alga, hook‐shaped appendages on cytoplasmic and spindle microtubules were detected. These resulted from the lateral association of a curved ribbon of protofilaments with the surface of a complete microtubular wall. In the fly, an S‐shaped protofilament sheet was found embedded in the kinetochore plate of a prometaphase I spematocyte. Tracing of the S‐shaped element towards the spindle pole revealed that it was formed by the lateral junction of two curved protofilament sheets. In all cases, the C‐shaped protofilament sheets represented the endings of complete micro‐tubules. Incomplete microtubules are generally considered as representing intermediates of microtubule assembly and disassembly. Since high molecular weight proteins are believed to be responsible for maintaining microtubule‐microtubule spacing, it is hypo‐thesized that the endings of growing and shrinking microtubules are sparsely studded with these proteins; their depletion allows lateral microtubule contacts. In addition, the microtubule‐microtubule contacts may be rendered possible by the flexibility of the slender elongated microtubule‐associated molecules. Relatively long C‐shaped proto‐filament appendages (0.6‐1.4 μm) were detected in this study. Therefore, it is plausible to assume that the protofilament sheets are stabilized by contact with one another or with an intact tubule wall.
Transmembrane cytoskeletal modulation in preterminal growing axons: I. Arrest of bulk and organelle transport in goldfish retinal ganglion cell axons regenerating in vitro by lectins binding to sialoglycoconjugates
Authors: Edmonds Brian T. , Koenig Edward
Published: February 2005

Goldfish retinal ganglion cell (RGC) axons regenerating in vitro exhibit a novel mode of axoplasmic transport that entails a rapid bidirectional bulk redistribution of axoplasm, “packaged” as protruding varicosities and non‐protruding phase‐dense inclusions (Koenig et al.: J. Neurosci. 5:715–729, 1985; Edmonds and Koenig Brain Res. 406:288–293, 1987). We have used phase‐contrast video microscopy to study transmembrane effects of surface‐binding lectins on bulk transport and transport of single visible organelles in RGC axons. Our findings show that certain lectins which crosslink sialoglycoconjugates, such as wheat germ agglutinin (WGA) and the more specific sialic acid‐binding lectin Limax flavus agglutinin (LFA), induce a rapid inhibition of transport activity. The LFA‐induced inhibition of transport can be reversed by appropriate simple sugar haptens, and can also be antagonized by pretreatment with cytochalasin D. One of the consequences of LFA binding is an increase in RITC‐conjugated phalloidin fluorescence staining of preterminal axons. The latter observation in conjunction with the antagonistic action of cytochalasin D suggests that one possible explanation for the transmembrane arrest of transport induced by crosslinking of surface sialoglycoconjugates may involve a polymerization and/or reorganization of the actin filament network which hinders translocation of mobile axoplasmic components.
Reorganization of circumferential microfilament bundles in retinal epithelial cells during mitosis
Authors: Sandig Martin , Kalnins Vitauts I.
Published: February 2005

To examine the behaviour of the apical circumferential microfilament bundles (CMBs) associated with the zonula adhaerens (ZA)‐junctions during mitosis, retinal pigment epithelial cells were labelled for F‐actin, and retinas were serially sectioned for TEM. The results show that the ZA‐CMB‐complex persists throughout all stages of mitosis. At metaphase, the cells round up, but stay joined apically to adjacent cells by ZA‐junctions. At telophase, the cleavage furrow forms asymmetrically from the basal end progressively toward the apical end, where the daughter cells remain connected by an intercellular bridge (IB). As the cleavage furrow with the contractile ring (CR) approaches the CMB, the two microfilament (MF) systems are oriented perpendicularly to each other. At the level of the CMB, the MFs of the CR connect the opposite sides of the CMB and bisect it into two CMBs, one for each of the two daughter cells. Subsequently, the CR in the IB splits into two, one on either side of the midbody. The two daughter cells, having acquired a complete CMB of their own, do not become direct neighbours, since adjacent cells, which remain joined to the apical ZA‐junction of the dividing cell, are observed in the cleavage furrow, where they meet and form a ZA‐junction between themselves, just below the IB. Separation of the daughter cells without losing contact with neighbouring cells at the level of the apical ZA‐junction thus maintains the integrity of the epithelial sheet during mitosis.
Dynamic properties of intermediate filaments: Disassembly and reassembly during mitosis in baby hamster kidney cells
Authors: Rosevear Ellen Rae , McReynolds Manette , Goldman Robert D.
Published: February 2005

A morphological analysis of the organizational changes in the type III intermediate filament (IF) system in dividing baby hamster kidney (BHK‐21) cells was carried out by immunofluorescence and immunoelectron microscopy. The most dramatic change occurred during prometaphase, when the typical network of long 10‐nm‐diameter IF characteristic of interphase cells disassembled into aggregates containing short 4–6 nm filaments. During anaphase‐telophase, arrays of short IF reappeared throughout the cytoplasm, and, in cytokinesis, the majority of IF were longer and concentrated in a juxtanuclear cap. These results demonstrate that the relatively stable IF cytoskeletal system of interphase cells is partitioned into daughter cells during mitosis by a process of disassembly and reassembly. This latter process occurs in a series of morphologically distinct steps at different stages of the mitotic process.
Myosin II antibodies inhibit the resorption activity of isolated rat osteoclasts
Authors: Sato Masahiko , Grasser William
Published: February 2005

We present microinjection data in support of an indirect approach by which cytoplasmic protein interactions important in the processes of bone resorption can be elucidated. Three polyclonal antibodies (M1, M3, M5) raised against myosin II from perfused rat liver differently affected the actin‐activated Mg ATPase of myosin II. These antibodies microinjected into isolated rat osteoclasts affected osteoclast morphology and activity in bone resorption. M1, which completely inhibited myosin ATPase activity at a antibody:myosin ratio of 10:1, initially promoted the extension/retraction motility of lamellipodia but eventually reduced the spread area of osteoclasts along the substrate after 20 hr. M3, which inhibited ATPase activity by 70%, had similar effects; however, M5, which weakly inhibited ATPase activity, neither promoted extension/retraction nor reduced spread area of osteoclasts. Immunofluorescence showed that these antibodies removed myosin II from the majority of actin filaments in injected osteoclasts. Because antibodies that did not bind to a myosin II column had little effect on the extension/retraction of lamellipodia or the osteoclast spread area, these data suggest that myosin II participates in the stabilization of osteoclast lamellipodia along the substrate. M1 injection strongly inhibited injected osteoclasts from excavating resorption lacunae in bone slices, compared to control antibody. M3 and M5 were less effective but also inhibited bone resorption. These data show that myosin II is functionally important in bone resorption and that the osteoclast‐differentiated activity of bone resorption is a more sensitive assay for myosin activity than lamellipodia motility or cell morphology.
Modulation of keratin intermediate filament distribution in vivo by induced changes in cyclic AMP‐dependent phosphorylation
Authors: Eckert Barry S. , Yeagle Philip L.
Published: February 2005

Treatment of PtK1 cells with 5 mM acrylamide for 4 hr induces reversible de‐phosphorylation of keratin in concert with reversible aggregation of intermediate filaments (Eckert and Yeagle, Cell Motil. Cytoskeleton 11:24–30, 1988). We have examined this phenomenon by 1) in vitro phosphorylation of isolated PtK1 keratin filaments and 2) combined treatments of PtK1 cells with both acrylamide and agents which elevate intracellular cAMP levels. PtK1 keratins were incubated in gamma‐32P‐ATP in the presence or absence of cAMP‐dependent kinase (A‐kinase) and cAMP. Levels of phosphorylation were analyzed by electrophoresis and autoradiography. Phosphorylation of keratin polypeptides (56 kD, 53 kD, 45 kD, 40 kD) occurred without added kinase, suggesting the presence of an endogenous kinase which remains with intermediate filaments in residues of Triton X‐100 extracted cells. Phosphorylation levels were increased by A‐kinase but not by cAMP alone, indicating the presence of cAMP‐dependent phosphorylation sites in addition to sites phosphorylated by the endogenous kinase. To study the possible role of cAMP‐dependent phosphorylation in acrylamide‐induced aggregation of keratin filaments, we treated cells with acrylamide in the presence of 8‐bromo‐cAMP (brcAMP), pertussis toxin (PT), isobutylmethylxanthine (IBMX), or forskolin, which increase intracellular cAMP levels. The distribution and phosphorylation levels of keratin filaments, as well as intracellular cAMP levels, were determined for each of these treatments. In addition to aggregation and dephosphorylation of keratin filaments reported previously, treatment of cells with acrylamide alone also results in reduced levels of intracellular cAMP. 8‐bromo‐cAMP, IBMX, and forskolin prevent acrylamide‐induced aggregation of keratin filaments and result in both normal levels of keratin phosphorylation and normal intracellular cAMP levels. PT was apparently ineffective. These observations suggest that 1) PtK1 keratins are phosphorylated by cAMP‐dependent kinase and an endogenous, cAMP‐independent kinase and 2) alteration of levels of cAMP‐dependent phosphorylation may be involved in aggregation of keratin filaments in response to acrylamide.
Localization of the centrin‐related 165,000‐Mr protein of PtK2 cells during the cell cycle
Authors: Baron Andre T. , Greenwood Tammy M. , Salisbury Jeffrey L.
Published: February 2005

In this study, we follow changes in localization of the centrin‐related 165,000‐Mr protein of PtK2 cells during the cell cycle. This protein is a component of a pericentriolar lattice that consists of pericentriolar satellites, pericentriolar matrix, and basal feet (Baron A.T., and J.L. Salisbury, J. Cell Biol. 107:2669–2678, 1988). By immunofluorescence microscopy, the 165,000‐Mr protein is seen as a constellation of pericentrosomal spots. We observe that cells in late G1 and S are characterized by a dense centrosomal focus of spots with additional spots dispersed throughout the cytoplasm. In G2, one bright centrosomal focus of clustered spots is observed. As the cells proceed through prophase this single focus divides, forming two foci that move toward opposite sides of the nucleus. During prometaphase, each polar focus of spots disperses. At metaphase, the spots are distributed throughout each half‐cytoplast from the poles to the chromosomes. During anaphase chromosome movement, some spots are seen beside and behind the trailing chromosome arms while others are clustered at the poles. At telo‐phase, pericentrosomal spots radiate from the poles to surround each mass of chromatin. In early G1, pericentrosomal spots surround each newly formed nucleus. We conclude that the 165,000‐Mr protein is a dynamic component of both the centrosome (pericentriolar matrix) and the mitotic apparatus (spindle matrix).
Microtubule reorganization during the cell cycle in synchronized BY‐2 tobacco suspensions
Authors: Hasezawa Seiichiro , Marc Jan , Palevitz Barry A.
Published: February 2005

Tobacco BY‐2 suspension cultures were synchronized with aphidicolin in order to assess the relationship between microtubules (MTs), microfilaments (MFs), and the nuclear envelope (NE) at different stages of the cell cycle. Using immunofluorescence techniques, ordered MT arrays were found in the cortex in G1; few MTs are evident deeper in the cytoplasm or near the nucleus. However, MTs radiate from the surface of the nucleus during S and G2 as the interphase cortical array is replaced by the preprophase band. Perinuclear fluorescence is also visible at the end of cytokinesis but does not overlap with new ordered cortical arrays early in G1. When isolated nuclei are examined, associated MTs are again evident in S and G2, but not in G1. Microfilaments are colocalized with the MTs in the radiating arrays, as ascertained by dual staining of cells with rhodamine phalloidin. Propyzamide treatment leads to the loss of MTs at all stages, while cytoplasmic and perinuclear MF networks persist. Conversely, cytochalasin D disrupts MFs, including those radiating from the nucleus during S and G2, without any apparent effect on MTs. The results cast doubt on a proposed role for the NE in the generation of cortical MTs in plants. A universal role for MFs in the deployment of MTs is also in question.
Effect of cytochalasins on actin in dividing root tip cells of Allium and Triticum: A comparative immunocytochemical study
Authors: McCurdy David W. , Palevitz Barry A. , Gunning Brian E. S.
Published: February 2005

A collaborative effort was initiated to resolve differences in two recent papers on the effects of cytochalasins in root cells. While both studies reported similar effects on interphase cells (i.e., replacement of microfilaments by many small specks and rods), Palevitz (Cell Motil. Cytoskeleton 9:283‐298, 1988) maintained that cytochalasins B and D induce actin aggregation at the poles of dividing Allium root cells at a concentration of 10 μM with rhodamine phalloidin as a reporter probe, whereas McCurdy and Gunning (Cell Motil. Cytoskeleton 15:76‐87, 1990) could not find these aggregates following antiactin immunocytochemistry in Triticum roots treated with CB at 50 μM. Employing identical methods and materials in the same laboratory, we found that CD induces polar actin aggregates in dividing cells of both species. However, the aggregates in Triticum are smaller and occur less frequently than those in Allium. A similar pattern is seen with CB.
Microvascular endothelial‐derived autacoids regulate pericyte contractility
Authors: Dodge Andrea B. , Hechtman Herbert B. , Shepro David
Published: February 2005

A silicone rubber assay is used in conjunction with morphometric measurements to characterize in vitro the contractile properties of retinal pericytes in response to endothelial secreted factors. Factor(s) present in conditioned media derived from pulmonary and retinal microvascular endothelial cells and pulmonary artery endothelial cells promote pericyte contractions. Using a radioimmunoassay significant levels of endothelin immunoreactivity are measured in conditioned media obtained from all three cell lines. Thrombin treatment enhanced endothelin‐like secretions by pulmonary microvascular endothelial cells, but significantly reduced levels of endothelin‐like immunoreactivity secreted by retinal microvascular endothelial cells. Synthetic endothelin and thromboxane A2 (TxA2) stimulate pericyte contractions, whereas prostaglandin I2 (PGI2) promotes pericyte relaxation. Thrombin and angiotensin II (ang II) have no effect on pericyte contractility. However, using cocultures of pericytes and endothelial cells we observe endothelial‐dependent pericyte contractions in response to thrombin and ang II. Thrombin and ang II stimulate the release of endothelial‐derived contracting factors, with characteristics similar to endothelin. These data suggest microvascular endothelial cell‐pericyte interactions may regulate, at least in part, microvessel contractility.
Variant cDNAs encoding proteins similar to the α subunit of chicken CapZ
Authors: Cooper John A. , Caldwell Jane E. , Gattermeir David J. , Torres Michelle A. , Amatruda James F. , Casella James F.
Published: February 2005

Chicken adult muscle and liver cDNA libraries were screened with a cDNA, α1, previously isolated from a chicken embryo library by screening with antibodies against the α subunit of chicken CapZ. cDNAs with a new coding region, called α2, were found in addition to ones with the α1 coding region. α2 predicts a protein sequence that matches exactly the N‐terminal sequence of 5 peptides prepared from CapZ α purified from chicken muscle, while the protein sequence predicted by α1 matches the peptides well, but not exactly. The predicted protein sequences of α1 and α2 are very similar to each other, and they are similar to those of the α subunit of capping protein from Dictyostelium [Hartmann et al., J. Biol. Chem. 163:5254–5254, 1989] and an actin‐binding protein from Xenopus [Ankenbauer et al., Nature 342:822–824, 1989]. Other conserved features of the predicted primary and secondary structures are noted. Chicken α1 and α2 are transcribed in all of 7 adult chicken muscle and non‐muscle tissues in comparable amounts by Northern analysis. α2 has four poly(A)+ RNA transcripts, one of which is rare in liver. α1 has two transcripts. α1 and α2 are encoded by different single‐copy genes by Southern analysis of chicken genomic DNA.
Decrease of internal free calcium and human sperm movement
Authors: Serres Catherine , Feneux Danielle , Berthon Brigitte
Published: February 2005

In order to elucidate the effects of calcium on the movement of human spermatozoa, studies were conducted using motile cells selected by swim‐up migration at 37°C in 5% CO2 in air in a synthetic BWW medium containing 1.7 × 10−3 M CaCl2 or BWW without added calcium (BWW‐Ca). Preliminary experiments have confirmed that the addition of EGTA (5 × 10−3; 10−2 M) to BWW medium decreased the intracellular calcium concentration ((Ca++)i) of spermatozoa, as measured in cells loaded with a fluorescent Ca++ indicator, Quin‐2. Concomitant measurements of (Ca++)i and sperm movement (analysed by videomicrography at 200 f/s at room temperature) were carried out on Quin‐2 loaded cells incubated in BWW‐Ca medium plus EGTA (10−5 M; 10−4 M; 10−3 M). Under these conditions a decrease in (Ca++)i was observed and associated with a decrease in mean amplitude of lateral head displacement (ALH). Analysis using an automatic analyser (Hamilton Thorn at 37°C) confirmed these results: the percentage of spermatozoa swimming with ALH ≤ 6 μm is decreased when the external free calcium in BWW‐Ca is decreased by the addition of 10−5 M, 10−4 M, or 10−3 M EGTA. Flagellar analysis of the sperm population characterized by ALH ≤ 6 μm showed a large proximal curvature of the tail associated with a low propagation wave velocity and a low beat frequency as compared to the spermatozoa with ALH ≤ 6 μm with similar progressive velocities. These characteristics result in a high flagellar beat efficiency (in terms of head displacement per beat). The disappearance of this pattern of movement when intracellular calcium is lowered indicates that calcium plays a complex role in the relationship between curvature and wave propagation. The ability of spermatozoa to modulate their movement in response to an alteration in the intracellular calcium level confirms the role of calcium in controlling flagellar movement in intact cells.
Relation of nucleolar structure and position to the cytoplasmic microtubule system in Dictyostelium
Authors: Sameshima Masazumi , Fujimoto Hirotaka , Imai Yoshihisa , Tsukita Shoichirou , Hashimoto Yohichi
Published: February 2005

The cytoplasmic microtubule system seems to influence the position and structure of nucleoli in Dictyostelium discoideum amoebae in several growing and migrating states. For example, nucleoli were usually excluded from the nuclear periphery near the microtubule‐organizing center (MTOC) in all cases; and in migrating adherent cells, more than half the nucleoli were located opposite the MTOC. This localization was disrupted by nocodazole treatment, after which the nucleoli were largely dispersed except near the MTOC. More extensive effects of microtubules on nucleolar structure were seen in aggregating cells. In contrast to the normal oval structure in growing cells, nucleoli took on a different morphology: they protruded from the leading edge of nuclei and elongated to form nozzle‐like structures. Analysis by rapid‐freeze substitution and indirect immunofluorescence showed each nozzle surrounded by more than 10 microtubules; and in the presence of nocodazole, the microtubules shortened as expected and the nozzles disappeared. Between microtubules and the outer nuclear envelope, various‐sized cross‐bridges were seen. The implication that microtubules were associated with the nucleoli in aggregating cells was verified in vitro: nuclei isolated from growing cells contained the MTOC but few if any detectable microtubules; but nuclei from aggregating cells were surrounded by them. These data are consistent with the notion the microtubule system may help regulate the position and conformation of nucleoli during early development of Dictyostelium.
Detection of spindle pushing forces in vivo during anaphase B in the fungus Nectria haematococca
Authors: Aist James R. , Bayles Carol J.
Published: February 2005

Forces that elongate the spindle during anaphase B of mitosis might be generated in the asteis, in the spindle, or in both. In the fungus Nectria haematococca, it has already been shown that the asters pull on the spindle pole bodies (SPBs) through‐out anaphase B. In this study, we used computerized video motion analysis to characterize brief episodes of spindle bending and straightening to find out if such bending is caused by spindle pushing forces. In three episodes there were two distinct components of spindle bending and straightening: one spanning the entire episode and comprising spindle elongation and another, superimposed on the first, involving a shortening of the distance between the SPBs. In a fourth episode, only spindle elongation was involved. All four spindles elongated rapidly while bending and underwent net growth during the overall bending‐straightening episode at an average rate of 4.2 μm/min. The path of one aster of a fifth mitotic apparatus was blocked by a large, occluding vacuole. This obstacle caused the migration of the mitotic apparatus to stop, resulting in a long (25 sec) episode of spindle curving and bending, usually without any substantial reduction in the distance between the SPBs as well as a marked reduction (from 4.7 to 0.65 μm/min) in the rate of spindle elongation. The results provide evidence that spindle pushing forces are active in vivo during anaphase B in N. haematococca and that they, along with astral pulling forces, help to elongate the spindle at a mostly constant rate. This is the first demonstration of both kinds of spindle elongation forces in the same organism.
A component of the interphase cytoskeleton is cyclically recruited into spindle poles during mitosis
Authors: Leslie Roger J. , Kohler Eric , Wilson Leslie
Published: February 2005

During the transition from interphase to mitosis, proteins are recruited into forming spindle poles [Leslie, Cell Motil. Cytoskeleton 16:225‐228, 1990]. Antibodies which recognize these recruited components clearly label spindle poles during mitosis but the location and character of such proteins during interphase remain a mystery. Competition assays using an antibody to a recruited spindle pole protein show that in its disperse form the spindle pole protein is a highly insoluble component of the Cytoskeleton which is dispersed to such an extent during interphase that it is difficult to identify by immunolocalization. The function of recruited spindle pole proteins is unknown but the aggregation/dispersion cycle and the antigen are highly conserved, appearing in sea urchin embryos and tissue culture cells.
An automated assay for quantifying the swimming behavior of Paramecium and its use to study cation responses
Authors: Clark Kevin D. , Nelson David L.
Published: February 2005

Paramecium letraurelia is a ciliated protist that alters its swimming behavior in response to various stimuli. Like the sensory responses of many organisms, these responses in Paramecium show adaptation to continued stimulation. For quantitative studies of the initial response to stimulation. and of the time course of adaptation, we have developed a computerized motion analysis assay that can detect deviations from the normal swimming pattern in a population of cells. The motion of an average of ten cells was quantified during periods ranging from 15 to 60 seconds, with a time resolution of 1/15 seconds. During normal forward swimming, the maximum deviation from a straight‐line path was less than 17°. Path deviations above this threshold value were defined as changes in swimming direction. The percentage of total path time that cells spent deviating from forward swimming was defined as percent directional changes (PDC). This parameter was used to construct dose‐response curves for the behavioral effects of various externally added cations known to induce behavioral changes and also to show the time course of adaptation to a depolarizing K+ stimulus. This assay is a valuable tool for studies of chemoeffectors or mutations that alter the swimming behavior of Paramecium and may also be applicable to other motile organisms.
Neural crest cell galvanotaxis: New data and a novel approach to the analysis of both galvanotaxis and chemotaxis
Authors: Gruler Hans , Nuccitelli Richard
Published: February 2005

The galvanotaxis response of neural crest cells that had migrated out of the neural tube of a 56‐hr‐old quail embryo, onto glass coverslips was observed using time‐lapse video microscopy. These cells exhibit a track velocity of about 7 μm/min and actively translocate toward the negative pole of an imposed DC electric field. This nonrandom migration could be detected for fields as low as 7 mV/mm (0.4 mV/cell lepgth). We find that this directional migration is independent of the speed of migration and have generated a rather simple mathematical equation that fits these data. We find that the number of cells that translocate at a given angle, Φ, with respect to the field is given by the equation N (Φ) = exp(a()0 + a1 cos Φ), where a1 is linearly proportional to the electric field strength for fields less than 390 mV/mm with a constant of proportionality equal to KG, the galvanotaxis constant. We show that KG = (150 mV/mm)−1, and at this field strength the cellular response is approximately half maximal. This approach to cellular translocation data analysis is generalizable to other directed movements such as chemotaxis and allows the direct comparison of different types of directed movements. This analysis requires that the response of every cell, rather than averages of cellular responses, is reported. Once an equation for N(Φ) is derived, several characteristics of the cellular response can be determined. Specifically, we describe (1) the critical field strength (390 mV/mm) below which the cellular response exhibits a simple, linear dependence on field strength (for larger field strengths, an inhibitory constant can be used to fit the data, suggesting that larger field strengths influence a second cellular target that inhibits the first); and (2) the amount of information the cell must obtain in order to generate the observed asymmetry in the translocation distribution (for a field strength of 100 mV/mm, 0.3 bits of information is required).