bluuebunny/crossref_metadata_2025 huggingface dataset:
Reactivation of isolated mitotic apparatus: Metaphase versus anaphase spindles
Authors: Palazzo Robert E. , Lutz Douglas A. , Rebhun Lionel I.
Published: February 2005

Mitotic spindles isolated from sea urchin eggs can be reactivated to undergo mitotic processes in vitro. Spindles incubated in reactivation media containing sea urchin tubulin and nucleotides undergo pole‐pole elongation similar to that observed in living cells during anaphase‐B. The in vitro behavior of spindles isolated during metaphase and anaphase are compared. Both metaphase and anaphase spindles undergo pole‐pole elongation with similar rates, but only in the presence of added tubulin. In contrast, metaphase but not anaphase spindles increase chromosome‐pole distance in the presence of exogenous tubulin, suggesting that in vitro, tubulin can be incorporated at the kinetochores of metaphase but not anaphase chromosomes. The rate of spindle elongation, ultimate length achieved, and the increase in chromosome‐pole distance for isolated metaphase spindles is related to the concentration of available tubulin. Pole‐pole elongation and chromosome‐pole elongation does not require added adenosine triphosphate (ATP). Guanosine triphosphate (GTP) will support all activities observed. Thus, the force generation mechanism for anaphase‐B in isolated sea urchin spindles is independent of added ATP, but dependent on the availability of tubulin. These results support the hypothesis that the mechanism of force generation for anaphase‐B is linked to the incorporation of tubulin into the mitotic apparatus. (If, in addition, a microtubule‐dependent motor‐protein(s) is acting to generate force, it does not appear to be dependant on ATP as the exclusive energy source).
Alterations in microtubule assembly caused by the microtubule‐active drug LY195448
Authors: Barlow Steven B. , Cabral Fernando
Published: February 2005

LY195448 is an experimental drug that blocks cells at metaphase (Boder et al.:Microtubules and Microtubule Inhibitors 1985: 353–361, 1985). A 4 hour exposure of NRK cells to a drug concentration of 46 μM (15 μg/ml) increased the number of mitotic cells in the population from 4.9% to 18.5%. Examination of treated cells by immunofluorescence showed increased numbers of cells blocked at prometaphase, with short microtubules extending from the spindle pole to the kinetochores. The cytoskeleton of interphase cells remained intact at these concentrations. However, the number of microtubules appeared to be reduced, and those that remained appeared kinkier and curled, particularly toward the periphery of the cells. When cytoskeletal microtubules of NRK cells were depolymerized with nocodazole, they reassembled within minutes of transfer to drug‐free media. However, nocodazole‐treated cells transferred to fresh media containing 15 μg/ml of LY 195448 required 2‐3 times longer to reassemble cytoplasmic microtubules. Previously isolated Chinese hamster ovary cell microtubule mutants resistant to either taxol or Colcemid were tested for cross‐resistance to this drug. Cell lines resistant to the depolymerizing drug Colcemid exhibited increased resistance to LY 195448 compared to wild‐type cells, whereas taxol resistant cell lines were more sensitive. Of eleven newly isolated mutant CHO cell lines selected for increased resistance to LY 195448, seven exhibited an altered β‐tubulin protein by two‐dimensional polyacrylamide gel electrophoresis. These 11 cell lines also showed a heterogenous pattern of resistance to several microtubule‐active drugs. These data demonstrate that LY 195448 is cytotoxic to mammalian cells because it inhibits microtubule assembly, most likely through a direct interaction with tubulin.
Effects of interferon‐β on the translocation rate and stationary time in human fibroblasts in culture
Authors: Murphy James S. , Tamm Igor
Published: February 2005

The rate of translocation and the percent of the time that cells are stationary have been measured by computer‐assisted time‐lapse cinemicrography in over 1,000 freshly planted human foreskin fibroblasts (FS‐4 cell strain) for periods of up to a week and the effects of interferon‐β (IFN‐β) on these parameters have been determined. Cells were planted at 2.5 × 103 cells/cm2 in Eagle's minimal essential‐medium (MEM) with 10% fetal bovine serum (FBS). Frames were taken every 2 or 4 minutes and data were collected on both cell location and cell division as a function of time. After planting FS‐4 cells require ∼48 hr to reach maximum motility both with respect to the translocation rate when moving and percent time cells are moving. Recombinant human IFN‐β (800 μ/ml) caused a marked increase in the fraction of time cells were stationary and a decrease of lesser magnitude in the translocation rate, as quantitated during the period during which the stationary fraction for control cells was at a minimum. IFN‐β also decreased the rate of cell proliferation, without any evidence of degeneration or death of cells. Our results contribute new evidence that the fraction of time cells spend moving directionally is an important determinant of their locomotory behavior and that this determinant is responsive to modulation by cytokines.
βVLDL uptake by pigeon monocyte‐derived macrophages: Correlation of binding dynamics with three‐dimensional ultrastructure
Authors: Jones Nancy L. , Lewis Jon C. , Allen Nina S.
Published: February 2005

Endocytosis of pigeon beta migrating very‐low‐density lipoprotein (βVLDL) by monocyte‐derived macrophages (monocyte/macrophages), cultured from Random Bred White Carneu (RBWC) pigeons, occurs by both coated and non‐coated regions of the plasma membrane (Henson et al.: Exp. Mol. Pathol. 51:243–263, 1989). Secondary to binding, the βVLDL is translocated to lysosomes for degradation. Ultimately these events lead to foam cell formation in vitro. Utilizing video‐enhanced contrast light microscopy in conjunction with whole mount intermediate‐voltage transmission electron microscopy (IVEM) and high‐resolution scanning EM, the dynamics of βVLDL binding have been correlated with ultrastructure. Beta VLDL conjugated to gold colloids was visualized at the surface of living cells by using Allen video‐enhanced contrast‐differential interference contrast microscopy (AVEC‐DIC). Subsequent to AVEC‐DIC, direct observation of the identical cells by IVEM and SEM was facilitated through the use of gold finder grids, and these EM observations confirmed identification of the videoobserved βVLDL particles.

Upon addition of βVLDL, pigeon monocyte/macrophages underwent gross morphological changes. These changes were recorded by video as movements at the cytoplasmic periphery, and the movements involved extension of microvilli, expression of retraction fibers, and elaboration of membrane ruffles. When secondarily observed by stereo (3‐D) IVEM and SEM, the identification of microvilli, retraction fibers, and membrane ruffles was confirmed and the lipoprotein‐gold conjugates were associated with these ligand‐induced membrane structures. Beta VLDL‐gold conjugates were also associated with pit‐like regions at the base of microvilli, while at the base of ruffles, βVLDL‐gold conjugates were located in membrane invaginations and cytoplasmic vesicles.
Pseudopodial activity at the active edge of migrating fibroblast is decreased after drug‐induced microtubule depolymerization
Authors: Bershadsky Alexander D. , Vaisberg Eugeni A. , Vasiliev Juri M.
Published: February 2005

It is known that depolymerization of microtubules by colcemid or other similar drugs abolishes polarization of pseudopodial activity in migrating fibroblasts. In this work the effect of colcemid on the intensity of protrusion and retraction of lamellipodia at the active edges of human fibroblasts migrating into the wound was investigated with video‐enhanced contrast microscopy. To characterize the pseudopodial activity quantitatively the outlines of the active edges in the pairs of frames taken at adjacent 20‐sec intervals were compared and mean areas of protrusions and retractions per unit length of the perimeter of the edge were measured. The mean rates of protrusions and retractions were 4–6 times less in colcemid‐treated cells than in controls. Thus, microtubules depolymerized by colcemid, and/or intermediate filaments undergoing perinuclear collapse in the presence of this drug, are essential not only for the restriction of pseudopodial activity to one particular zone of the cell edge but also for the development of maximal activity in this zone.
Segregation of the nucleolus during mitosis in budding and fission yeast
Authors: Granot David , Snyder Michael
Published: February 2005

The segregation of the nucleolus during mitosis was examined in Saccharomyces cerevisiae and Schizosaccharomyces pombe by indirect immunofluorescence using antibodies directed to highly conserved anti‐nucleolus antigens. In mitotic S. pombe cells, the nucleolus appears to trail the bulk of the DNA. In wild‐type cells of S. cerevisiae, the nucleolus segregates alongside the bulk of the genomic DNA. Based on its distance from the centromere, we would expect the rDNA in both organisms to segregate behind the majority of the genomic DNA, if telomeric regions trail centromeric regions as in other eukaryotes. We therefore suggest that in S. cerevisiae the nucleolus is attached to other parts of the nucleus which enable it to segregate along with the bulk of the DNA. The segregation of the nucleolus in topoisomerase mutants and nuclear division mutants of S. cerevisiae was also investigated. In cdc14 mutants which arrest at late anaphase, the vast majority of the DNA is separated, but the nucleolar antigens remain extended between the mother and daughter cells. Thus, the CDC14 gene of S. cerevisiae appears to be important for the separation of the nucleolus at mitosis.
Vinculin in relation to stress fibers in spread platelets
Authors: Nachmias V. T. , Golla R.
Published: February 2005

To investigate the function of vinculin in blood platelets, we studied its localization in relation to other cytoskeletal proteins as well as its state of phosphorylation in platelets allowed to spread on fibrinogen‐coated surfaces. By 5 minutes after loading the platelets onto the surfaces the 47 and 20 kDa polypeptides became phosphorylated, indicating activation. By 30 minutes, platelets formed small, typical bundles of fibers which stained brilliantly with rhodamine phalloidin. Myosin and tropomyosin, detected with specific antibodies, were localized in periodic arrays along these bundles. By indirect immunofluorescence, a discrete patch of vinculin was observed at each end of every actin‐containing bundle. Vinculin phosphorylation was not detected in immunoprecipitates protected against phosphatases. Interference reflection images showed that regions of close binding to the substratum (adhesion plaques) closely matched the vinculin staining sites. Talin appeared diffusely localized. It could be shown to be present in the plaques when platelets were stabilized with ZnCl2 by the method of Geiger and then sonicated to remove some of the surface membrane. Localizations of vinculin and myosin were unaltered by this treatment. Talin phosphorylation or proteolysis could not account for vinculin translocation.

We conclude that platelets, in response to an appropriate physiological surface, form typical actin bundles with vinculin at the termination of each bundle, in close relation to adhesion plaques. The signal for this translocation does not appear to depend on phosphorylation of vinculin or on phosphorylation or proteolysis of talin. Our findings support the conclusion that in platelets, as in nucleated cells, vinculin serves as at least part of the connection between bundled actin fibers and the extracellular matrix. Such a connection seems required for platelets' known ability to exert tension on surfaces.
Dynamic morphology of metastatic mouse T‐lymphoma cells invading through monolayers of 10T½ cells
Authors: Verschueren Hendrik , De Baetselier Patrick , Bereiter‐Hahn Jürgen
Published: February 2005

We have used an in vitro model system to analyze cytomechanical aspects of tissue infiltration by T‐lymphocytes. The interaction of metastatic T‐lymphoma cells with a precultured monolayer of 10T½ fibroblast‐like cells was recorded in time‐lapse video with alternating phase contrast and reflection interference contrast microscopy. Sectioning of embedded specimens as well as cytoskeletal stainings have been performed on matching cocultures.

The lymphoma cells did not strongly attach or spread on the dorsal surface of the monolayer cells. Invasion started with the protrusion of a pseudopodium through a narrow gap, and conspicious constriction of the invading cell's body and nucleus was a consistent feature during the later steps. Overt retraction of the target cells was not seen, but the invading lymphoma cells elevated the fibroblasts over relatively large areas, thereby creating dome‐shaped open spaces, allowing for further migration under the monolayer with minimal resistance. Invasion was not unidirectional but was readily reversible at any stage. Due to this wavering character, an invasion event could take more than 1 hour, although the shape alterations involved were fast. Even after the invasion process had been completed, the lymphoma cells could come out from below the monolayer again. Therefore we propose that invasion in this model should be considered as a dynamic equilibrium.

Invading T‐lymphoma cells displayed diffuse F‐actin staining and a well‐organized microtubular complex with the centrosomes behind the nucleus in the uropod, which also contained most vesicular organelles.
The redeployment of F‐actin in silk glands during moulting
Authors: Henderson Scott C. , Locke Michael
Published: February 2005

Labeling of silk glands with rhodaminyl‐phalloin shows that most F‐actin is restricted to parallel bundles that form rings around the gland lumen at the apical cell surface. The bundles are lost when larval feeding stops at moulting, and the F‐actin is redistributed through the cytoplasm as coats to vacuoles and, occasionally, in variably oriented strands. After moulting there is a return to the distribution of filamentous actin in the apical periluminal rings of bundles. These events occur at the same time as F‐actin in the nuclear shell [Henderson and Locke, submitted] undergoes its own set of changes. In silk gland cells two kinds of f‐actin deployment take place concurrently.
Incorporation of fluorescently labeled contractile proteins into freshly isolated living adult cardiac myocytes
Authors: Lorusso Stephen M. , Imanaka‐Yoshida Kyoko , Shuman Henry , Sanger Jean M. , Sanger Joseph W.
Published: February 2005

When fluorescently labeled contractile proteins are injected into embryonic muscle cells, they become incorporated into the cells' myofibrils. In order to determine if this exchange of proteins is unique to the embryonic stage of development, we isolated adult cardiac myocytes and microinjected them with fluorescently labeled actin, myosin light chains, α‐actinin, and vinculin. Each of these proteins was incorporated into the adult cardiomyocytes and was colocalized with the cells'native proteins, despite the fact that the labeled proteins were prepared from noncardiac tissues. Within 10 min of injection, α‐actinin was incorporated into Z‐bands surrounding the site of injection. Similarly, 30 sec after injection, actin was incorporated into the entire I‐bands at the site of injection. Following a 3‐h incubation, increased actin fluorescence was noted at the intercalated disc. Vinculin exchange was seen in the intercalated discs, as well as in the Z‐bands throug hout the cells. Myosin light chains required 4–6 h after injection to become incorporated into the A‐bands of the adult muscle. Nonspecific proteins, such as fluorescent BSA, showed no association with the myofibrils or the former intercalated discs. When adult cells were maintained in culture for 10 days, they retain the ability to incorporate these contractile proteins into their myofibrils. T‐tubules and the sarcoplasmic reticulum could be detected in periodic arrays in the freshly isolated cells using the membrane dye WW781 and DiOC3[3], respectively. In conclusion, the myofibrils in adult, as in embryonic, muscle cells are dynamic structures, permitting isoform transitions without dismantling of the myofibrils.
Cytokinesis is more rapid in Ha‐T24‐ras transfected rat embryo fibroblasts than in non‐transfected control cells
Authors: Ng Grace , Boylan John , Zimmer Stephen G. , Sisken Jesse E.
Published: February 2005

It has long been known that neoplastic cells are characterized by increases in cell motility. Earlier studies from this laboratory indicated that rnitotic events were also altered in many tumor and experimentally transformed cells and that this included increases in metaphase duration and a reduction in the duration of cytokinesis. The studies presented in this paper were done to determine whether or not transfection of normal rat embryo fibroblasts by the Ha‐T24‐ras oncogene could also produce such alterations in mitotic events. The results obtained with the use of time lapse video microscopy indicate that neither the duration of metaphase nor the rate of chromosome movement during anaphase was altered but that the rate of furrow progression during cytokinesis occurred at a significantly more rapid rate. Thus, the cellular alteratioons induced by transfection with Ha‐T24‐ras accelerate microfilament‐dependent cytokinetic furrowing without significant effects on microtubule‐dependent mitotic events. One of several possible mechanisms that could account for these observations involves a down regulation of protein kinase C which has been reported to occur in many neoplastic cells including those transformed by ras. Such a hypothesis could also have broader implications because it may be applicable to the increase in motility and metastatic activity generally observed in transformed cells.
Reversible intracellular ATP changes in intact rat spermatozoa and effects on flagellar sperm movement
Authors: Jeulin Claudette , Soufir Jean‐Claude
Published: February 2005

The initiation of motility and modification of energy metabolism of rat caudal epididymal spermatozoa can be induced by dilution in a saline medium. We have investigated in these cells the relationships between the energy reserve (sperm ATP content measured by bioluminescence) and flagellar movement (high speed videomicrography, 200 frames/sec). A steady state was observed in sperm ATP content, progressive velocity (Vp) and flagellar beat frequence (F) with sperm dilution in a medium with glucose, lactate, pyruvate and acetate substrates after 30 minutes of incubation, without these substrates, changes in metabolic pathways occurred immediately and initially disturbed the relationship between ATP levels and F, suggesting differences in motility initiation when energy is from an endogenous origin via mitochondrial oxidative phosphorylation. This “energy crisis” was reversed by the addition of substrates to the medium.

The three‐dimensional flagellar movement observed in the presence of substrates quickly became two‐dimensional in their absence. The flagellar beat envelope became more splayed, the mean amplitude of lateral head displacement increased and F decreased. The resulting high flagellar beat efficiency can be compared to that observed during hyperactivation which is a physiological event related to a fall in intracellular ATP level. In both media, the displacement of the flagellum in relation to the wave axis varied sinusoidally. The sine period increased with time when the spermatozoa were incubated in the medium without substrates. These results suggest a gradual slowing‐down of the velocity of wave formation in the proximal part of the flagellum.
Actin‐dependent myoid elongation in teleost rod inner/outer segments occurs in the absence of net actin polymerization
Authors: Pagh‐Roehl Kathryn , Brandenburger J. , Wang E. , Burnside B.
Published: February 2005

In the retinas of teleost fish, rod photoreceptors elongate in response to light. Light‐activated elongation is mediated by the myoid of the rod inner segment and is actin‐dependent. Inner segment F‐actin filaments form bundles running parallel to the cell's long axis. We examined the mechanism of rod elongation using mechanically‐detached rod fragments, consisting of the motile inner segment and sensory outer segment (RIS‐ROS). When RIS‐ROS are isolated from darkadapted green sunfish and cultured in the light, they elongate 15μm at 0.3–0.6μm/min. Elongation was inhibited 65% by 0.1μM Cytochalasin D, suggesting a requirement for actin assembly. To determine the extent of assembly during elongation, we used three approaches to measure the F‐actin content in RIS‐ROS: detection of pelletable actin by SDS‐PAGE after detergent‐extraction of RIS‐ROS; quantification of fluorescein‐phalloidin binding by fluorimetry, fluorescence‐activated cell sorting and image analysis; estimation of total F‐actin filament length by electron microscopy. All three assays indicated that no net assembly of RIS‐ROS F‐actin accompanied myoid elongation. An increase in F‐actin content within the elongated myoid was counterbalanced by a decrease in F‐actin content within the 13 microvillus‐like calycal processes located at the end of the inner segment opposite to the growing myoid. O'Connor and Burnside (Journal of Cell Biology 89:517–524, 1981) showed that minus‐ends of rod F‐actin filaments are oriented towards the elongating myoid while plus‐ends are oriented towards the shortening calycal processes. Our observations suggest that RIS‐ROS elongation entails actin polymerization at the minus‐ends of filaments coupled with depolymerization at the filament plus‐ends.
Two distinct isoforms of sea urchin egg dynein
Authors: Grissom Paula M. , Porter Mary E. , McIntosh J. Richard
Published: February 2005

Extracts of unfertilized sea urchin eggs contain at least two isoforms of cytoplasmic dynein. One exhibits a weak affinity for microtubules and is primarily soluble. The other isoform, HMr‐3, binds to microtubules in an ATP‐sensitive manner, but is immunologically distinct from the soluble egg dynein (Porter et al.: Journal of Biological Chemistry 263:6759–6771, 1988). We have now further distinguished these egg dynein isoforms based on differences in NTPase activity. HMr‐3 copurifies with NTPase activity, but it hydrolyzes CTP at 10 times the rate of ATP. The soluble egg dynein is similar to flagellar dynein in its nucleotide specificity; its MgCTPase activity is ca. 60% of its MgATPase activity. Non‐ionic detergents and salt activate the MgATPase activities of both enzymes relative to their MgCTPase activities, but this effect is more pronounced for the soluble egg dynein than for HMr‐3. Sucrose gradient‐purified HMr‐3 promotes an ATP‐sensitive microtubule bundling, as seen with darkfield optics. We have also isolated a 20 S microtubule translocating activity by sucrose gradient fractionation of egg extracts, followed by microtubule affinity and ATP release. This 20 S fraction, which contains the HMr‐3 isoform, induces a microtubule gliding activity that is distinct from kinesin. Our observations suggest that soluble dynein resembles axonemal dynein, but that HMr‐3 is related to the dynein‐like enzymes isolated from a variety of cell types and may represent the cytoplasmic dynein of sea urchin eggs.
Intracellular cyclic AMP produces effects opposite to those of cyclic GMP and calcium on shape and motility of neuroblastoma cells
Authors: Bolsover Stephen R. , Gilbert Susan H. , Spector Ilan
Published: February 2005

We have directly evaluated the effects of various intracellular second messengers including cyclic nucleotides, calcium ion, and inositol polyphosphates on shape and motility of differentiating mouse neuroblastoma cells. The messengers were microinjected into cells and the responses of the soma, neurite, and growth cone were monitored using time‐lapse video microscopy. Each messenger altered cell shape and motility in a characteristic manner. Cyclic AMP promoted lamellipodial expansion, neurite outgrowth, and motility. The other injected messengers opposed motility. Cyclic GMP caused motile structures to freeze and to retract permanently, while the inhibitory effects of calcium injection were concentrationdependent. Small calcium injections affected specifically actincontaining motile structures which froze and retracted temporarily. Intermediate calcium injections caused a strong contraction at the site of injection in all cells. With large injections, cells retracted long neurites, rounded up, and frequently began vigorous blebbing that continued to cell death. Injections of the inositol polyphosphates 1P3(1,4,5) and IP4(1,4,5,6) mimicked the effects of small calcium injections, as did electrical stimulation that elicited action potentials. The results suggest that in mouse neuroblastoma cells, intracellular CAMP elevation increases cytoskeletal organization and promotes neurite extension perhaps through an enhancement of cell‐substratum adhesion. On the other hand, a rise of intracellular cGMP or intracellular calcium interferes directly with the function and organization of the actin‐microfilament system. The integrated action of these second messenger systems may, therefore, operate in vivo to allow substances released from neighboring cells to regulate neuronal architecture. © 1992 Wiley‐Liss, Inc.
Overexpression of vinculin suppresses cell motility in BALB/c 3T3 cells
Authors: Fernández José Luis Rodríguez , Geiger Benjamin , Salomon Daniela , Ben‐Ze'ev Avri
Published: February 2005

The content of vinculin, a cytoplasmic protein found in focal contacts and cell‐cell junctions, was increased in BALB/c 3T3 cells by gene transfection. The vinculin expressed from the full length chicken cDNA, incorporated into focal contacts and its pattern was identical to that of the endogenous protein. Cells stably expressing vinculin by 20% over the endogenous level had altered locomotory properties. In these cells, the ability to migrate into a wound formed in a confluent monolayer and the locomotion of individual cells were drastically reduced. The results provide direct evidence that cell locomotion can be regulated by modulating vinculin expression. © 1992 Wiley‐Liss, Inc.
Immunohistochemical studies on the distribution of cellular myosin II isoforms in brain and aorta
Authors: Murakami Noriko , Elzinga Marshall
Published: February 2005

The distribution of nonmuscle myosin isoforms in brain and aorta was studied by using polyclonal antibodies against two synthetic peptides selected from a region near the carboxyl terminus of bovine brain (peptide IIB) and human macrophage (peptide IIA) myosin. Immunoblots of brain homogenates and purified myosin showed two major bands stained by anti‐peptide IIB (MIIB1 and MIIB2) and a minor band stained by anti‐peptide IIA (MIIA2). Polyclonal anti‐human platelet myosin antibodies did not react with MIIB isoforms. In cryosections from bovine, rat, and mouse brains, anti‐peptide IIB stained most neuronal cells. In bovine cryosections, glial staining was also observed. In contrast, anti‐peptide IIA and anti‐platelet myosin antibodies primarily stained blood vessels. In bovine aorta, the anti‐peptide antibodies recognized four bands, MIIB3, MIIB4, MIIA1, and MIIA2. Only MIIA2 was recognized by anti‐human platelet myosin antibodies. In bovine aorta cryosections, anti‐peptide IIB stained smooth muscle cells in tunica intima and tunica media but did not stain endothelial cells. Anti‐peptide IIA stained smooth muscle cells in the tunica media, and endothelial cells of vaso vasorum but not of aorta. Only polyclonal anti‐platelet myosin antibodies stained the endothelial cells of aorta tunica intima. These results indicate that multiple isoforms of cellular myosins exist in mammals, that these isoforms are expressed in a cell specific manner, and that the major myosin isoforms isolated from whole brain originate from neurons and, at least in bovine brain, from glia, but not from blood vessels. © 1992 Wiley‐Liss, Inc.
Mechanics of the chloroplast rotation in Mougeotia: Measurement of angular velocity by laser diffractometry
Authors: Ytow Nozomi , Yamada Toru , Ishizaka Shozo
Published: February 2005

The cell of the green alga Mougeotia orients its chloroplast by rotation, according to the direction or polarization of incident red light. The mechanics of the rotation is described by the angle of rotation and the angular velocity of the rotator (i.e., the chloroplast). We developed a laser diffractometer to determine the angle of rotation of the chloroplast. The angle of rotation of the chloroplast shifted by a constant angular velocity, and hence, the net torque on the chloroplast was zero. This suggests that the driving torque acting on the chloroplast is always balanced by the viscous torque. The maximal driving force acting on the chloroplast was estimated to be nearly equal to the force generated by an actomyosin system. This is the first measurement of the driving force acting on the chloroplast in Mougeotia. The amplitude of the force supported the anchorage site hypothesis. However, it remains unclear whether or not the angular independence of the force also supports the hypothesis. © 1992 Wiley‐Liss, Inc.
240 kDa immunoanalogue of vertebrate α‐spectrin occurs in Paramecium cells
Authors: Kwiatkowska Katarzyna , Sobota Andrzej
Published: February 2005

Polypeptide of 240 kDa immunorelated to vertebrate α‐spectrin was detected in Paramecium cells. The antigen was identified by monospecific antibody directed against α‐subunit of spectrin, which was isolated from chicken erythrocytes by affinity and anion‐exchange chromatography. Immunoblotting tests demonstrated that the anti‐α‐spectrin cross reacted with the 240 kDa polypeptide of Paramecium as well as that of various vertebrate cells. In Paramecium, the antigen was detected in cytoskeletal fraction and in contractile extract of the cells. Immuno‐fluorescent and immunoelectron microscopy observations revealed cortical localization of α‐spectrin immunoanalogue in Paramecium. The label was distinctly seen on the whole surface of trichocyst tips. The antigen was also distributed close to the inner alveolar membrane forming a regular, continuous, lattice‐like structure. When stained with rhodamine‐phalloidin, Paramecium cells displayed a similar fluorescent network, which underlay contours of cortical units. Basal bodies of cilia were labeled with phalloidin as well. Detection of α‐spectrin immunoanalogue in Paramecium cortex may provide a new insight into arrangement of cortical elements in this organism. © 1992 Wiley‐Liss, Inc.
A shell of F‐actin surrounds the branched nuclei of silk gland cells
Authors: Henderson Scott C. , Locke Michael
Published: February 2005

The branched nuclei from silk gland cells of larvae of Calpodes ethlius label with antibodies to actin and myosin and with rhodaminyl‐phalloin, which is specific for f‐actin. Optical sectioning localizes this actin and myosin to the nuclear periphery. Residual nuclear‐associated fractions prepared from these cells contain sheets of nuclear lamina‐like structures that bind heavy meromyosin and gold‐tagged antibodies to actin and myosin. The results suggest that both actin and myosin, or a myosin‐like protein, are components of a layer at the nucleocytoplasmic boundary that we call the nuclear shell. The nuclear shell appears to be associated with the nuclear envelope and may correspond to a zone on the cytoplasmic face of the envelope seen in electron micrographs of unextracted cells. The residual nuclear‐associated fraction has a unique isoform of actin (43 kD, pl 6.45) that might allow the nuclei to associate with an actin network structurally and developmentally distinct from that of the cytoplasm. © 1992 Wiley‐Liss, Inc.
Immunological comparison of 22S, 19S, and 12S dyneins from Paramecium cilia
Authors: Walczak Claire E. , Marchese‐Ragona Silvio P. , Nelson David L.
Published: February 2005

Three forms of dynein (22S, 19S, and 12S) were purified from Paramecium cilia. Two classes of monoclonal antibodies against purified 22S dynein were generated. One class reacted on immunoblots with the heavy chains of 22S, 19S, and 12S dyneins; the second class reacted with an 88 kD intermediate chain of 22S dynein. Polyclonal antiserum to the heavy chains of 22S dynein reacted with the γ‐heavy chain of 22S and 19S dyneins. A previously described antiserum raised against 22S dynein [Travis et al.: Biochim. Biophys. Acta 966:73–83, 1988] recognized the γ‐heavy chain of 22S dynein which was also present in 19S and 12S dyneins, along with the 88 and 76 kD intermediate chains of 22S dynein. This antiserum was also able to immunoprecipitate dynein from crude extracts of cilia.

Electron microscopy revealed that the 22S dynein consisted mainly of two‐headed particles with some three‐headed particles present. The 12S dynein was mainly one‐headed particles. The 19S dynein was a mixture of three‐, two‐, and one‐headed particles. The immunological and electron microscopic studies showed that 19S dynein arises from 22S dynein, and that 12S dynein is heterogeneous, composed of the γ‐heavy chain of 22S dynein and a unique dynein ATPase.

The polyclonal antibodies were also used to detect cross‐reactive proteins in other organisms. Both the anti‐heavy chain and the anti‐22S dynein sera reacted strongly with 22S outer arm dynein of Tetrahymena, but not with the 14S dynein of this organism. © 1993 Wiley‐Liss, Inc.
Ciliary dynein of Paramecium tetraurelia: Photolytic maps of the three heavy chains
Authors: Beckwith Susan M. , Asai David J.
Published: February 2005

The ciliate Paramecium tetraurelia presents a powerful system to define the structural basis for dynein functional diversity within a single cell. This analysis will depend on the biochemical resolution of the dynein proteins. As an important first step, the three heavy chains of the ciliary outer arm dynein of paramecium were characterized. Sucrose density gradient centrifugation in a high salt buffer separated the dynein into a 22S species, which contained the α and β heavy chains, and a 12S species, which contained the α chain as well as the inner arm dynein heavy chains. Both the 22S and 12S species retained enzymatic latency as indicated by stimulation of MgATPase activity by 0.1% Triton X‐100. An unusual ATP‐independent V1‐like photolysis of only the β chain provided the basis for estimating that the β chain contributes almost half of the 22S MgATPase activity that is susceptible to V1 photolysis. The combination of the density gradient separation of the partially dissociated dynein and the ATP‐independent V1‐like photolysis of only the β chain led to the unambiguous assignment of the V1 photolytic products to the appropriate parent heavy chains. An estimate of the molecular sizes of the three heavy chains was obtained. The photolytic peptide maps, which define the ATP‐binding domains, were determined for the three heavy chains. © 1993 Wiley‐Liss, Inc.
Divergent neural β tubulin from the antarctic fish notothenia coriiceps neglecta: Potential sequence contributions to cold adaptation of microtubule assembly
Authors: Detrich H. William , Parker Sandra K.
Published: February 2005

The cytoplasmic microtubules of the cold‐adapted Antarctic fishes, unlike those of homeotherms and temperate poikilotherms, assemble and function at body temperatures in the range −1.8 to +2°C. To determine whether alterations to the primary sequence of β tubulin may contribute to enhancement of microtubule assembly at cold temperatures, we have cloned and sequenced a 1.8‐kilobase neural β‐chain cDNA, Ncnβ1, from an Antarctic rockcod, Notothenia coriiceps neglecta. Based on nucleotide sequence homology, Ncnβ1 probably corresponds to a class‐II β‐tubulin gene. The 446‐residue β chain encoded by Ncnβ1 is closely related (sequence homology ∼95%) both to the neural class‐I/II isotypes and to the neural/testicular class‐IV variants of higher vertebrates, but the sequence of its carboxy‐terminal isotype‐defining region (residues 431–446) has diverged markedly (≥ 25% change relative to the I/II/IV referents). Furthermore, the NcnβsZ1 polypeptide contains six unique amino‐acid substitutions (five conservative, one nonconservative) not found in other vertebrate brain isotypes, and the carboxyterminal region possesses a unique tyrosine inserted at position 442. We conclude that Ncnβ1 encodes a class‐II β tubulin that contains sequence modifications, located largely in its interdimer contact domain, that may contribute to cold adaptation of microtubule assembly. © 1993 Wiley‐Liss, Inc.
Centripetal movement of homologs occurs at the onset of anaphase A in primary oocytes of Ephestia kuehniella Z. (Pyralidae, Lepidoptera)
Authors: Wolf Klaus Werner
Published: February 2005

The transition from metaphase to anaphase was examined in primary oocytes of the Mediterranean mealmoth, Ephestia kuehniella. Isolated spindles were analysed using phase‐contrast microscopy and antitubulin immunofluorescence. Metaphase I bivalents in female Lepidoptera contain material derived from the synaptonemal complexes, the elimination chromatin, which is located between the homologs. Upon the onset of anaphase I, the homologs detach from the elimination chromatin. The present observations revealed that prior to the start of anaphase A, the homologs move centripetally, i.e., toward the central spindle axis, while the elimination chromatin remains stationary. This type of movement is not directly comparable with conventional chromosome movements such as prometaphase congression, anaphase A, and anaphase B, since it occurs at right angles to the preferential orientation of the spindle MTs. A MT‐based mechanism may nevertheless account for the centripetal shift of homologs in meiosis I of the moth: bundling of kinetochore MTs of different chromosomes could transfer the chromosomes toward the spindle axis. Since the homologs move along the poleward surfaces of the plate of elimination chromatin, a MT‐independent force producer could exist as well at the interface between the chromosomes and the plate of elimination chromatin. © 1993 Wiley‐Liss, Inc.
Reactivation of demembranated, cytosol‐free ram spermatozoa
Authors: San Agustin Jovenal T. , Witman George B.
Published: February 2005

A procedure for preparing cytosol‐free ram sperm models was developed. Sperm are introduced to a Triton X‐100‐containing demembranation medium layered on top of a discontinuous Percoll gradient. After brief exposure to the demembranating solution, the sperm are separated from the detergent‐soluble components by centrifugation through a 55% Percoll layer, finally collecting on top of a 90% Percoll cushion from where they are recovered. Optimum conditions consisted of Triton X‐100 at 0.20% and a demembranation time of 35 sec. Cross‐sections of midpieces and principal pieces of the demembranated sperm were examined by electron microscopy. With 0.20% Triton X‐100 in the demembranation medium, 86% of the cross‐sections showed no plasma membranes and the rest had broken plasma membranes. The remaining tail structures appeared to be morphologically intact. Assay of phosphoglucose isomerase as a marker enzyme confirmed that at least 98% of the cytosolic protein was removed. Ram sperm models obtained by this procedure could be reactivated, and the percent motility and beat parameters were similar to those of the intact sperm. Reconstitution with the detergent‐soluble components was neither required for, nor enhanced, reactivation. Therefore, demembranated ram sperm do not require a detergent‐soluble protein factor for reactivation. © 1993 Wiley‐Liss, Inc.
Colchicine‐induced stimulation of PMN motility related to cytoskeletal changes in actin, α‐actinin, and myosin
Authors: Keller H. U. , Niggli V.
Published: February 2005

Colchicine‐induced stimulation of polymorphonuclear leukocyte (PMN) locomotion is an interesting model because extension of blebs at the front occurs at a rate (about 2.4 μm/s) which is far above that reported for growth of actin filaments. The following cytoskeletal changes were observed in colchicine‐treated PMNs: (1)a small increase in cytoskeleton‐associated actin was noted, as well as a somewhate more pronounced increase in cytoskeleton‐associated α‐actinin, as compared with untreated or DMSO‐treated controls. There was, however, no measurable increase in F‐actin as determined by NBD‐phallacidin blinding; (2)the values for the ratio (α‐actinin/actin) are lower in PMNs treated with colchicine for 30 min, as compared with PMNs stimulated with fNLPNTL for 1 minute (non‐polar ruffling cells) or 30 min (polarized locomoting cells); thus, this ratio may depend on the type of PMN motility; (3) in polarized PMNs F‐actin was mainly located linearly all along the cell membrane; there was more intense staining at the front of the cells; (4) α‐actining appeared to colocalize with F‐actin at the leading front, but not with F‐actin at the tail of polarized cells; (5) myosin was preferentially found at the rear part of polarized cells but not or only to a small extent at the front. Our data indicate a close functional correlation between microtubules and microfilaments. We speculate that F‐actin in combination with α‐actinin promotes expansion of pseudopods, whereas myosin combined with F‐actin promotes contraction. In more general terms we suggest that different forms of PMN motility are generated by differential selective interaction of cytoskeletal compnents and variations in the composition of the cytoskeleton in different sites of the same cells. © 1993 Wiley‐Liss, Inc.
βIV is the major β‐tubulin isotype in bovine cilia
Authors: Renthal Robert , Schneider Barbara G. , Miller Margaret M. , Ludueña Richard F.
Published: February 2005

Four different isotypes of β‐tubulin are known to be expressed in mammalian brain. Monoclonal antibodies against βII, βIII, and βIV were used to characterize the β‐tubulin isotypes in two ciliated bovine tissues: non‐motile sensory cilia of retinal rod cells and motile cilia of tracheal epithelium. Retinal rod outer segment (ROS) connecting cilia and cytoskeletons were purified by density gradient centrifugation. This preparation contained more than 20 major protein protein components, as shown by dodecyl sulfate polyacrylamide gel electrophoresis. Electroblots were used to quantitate the relative amounts of βII, βIII, and βIV. The connecting cilium and cytoskeleton of the rod outer segment has less type III β‐tubulin than brain and more type IV. The ratio of βIV to βII in the ROS is nearly a factor of 8 larger than in brain. Electron microscopic immunocytochemistry showed extensive labeling of cilia by anti‐type IV in thin sections of retinas and trachea, and also in purified ROS cilia and cyoskeletons. Labeling of cilia by anti‐βII was also observed, although in the purified ROS cilia and cytoskeleton, the anti‐βII labeling was primarily on amorphous non‐ciliary material. The results suggest that both motile and non‐motile cilia are enriched in the type IV β‐tubulin subunit. © 1993 Wiley‐Liss, Inc.
Analysis of the elongation of the coiled filamentous structure, the truncated cone, during the acrosome reaction of abalone sperm by computer simulation and quick‐freeze, deep‐etch electron microscopy
Authors: Sakai Yoshi T. , Shinagawa Yoshihisa , Shiroya Yoko , Kunii Tosiyasu L.
Published: February 2005

A novel structure named the truncated cone was located in the apex of the acrosomal vesicle right beneath the outer acrosomal membrane of abalone sperm head. This truncated cone structure was composed of about 12 helically coiled filaments, each 3.5–3.6 μm long with a diameter of 8–12 nm, forming a tightly compressed helicoid. During the acrosome reaction, the truncated cone elongated more than three times the original height and transformed into a thin cylinder by further coiling up of the filaments from the initial 2.5 to final 5 turns. The diameter and the lenght of each filament did not change during the elongation of the truncated cone into thin cylinder.

Calculation from the equation of helical movement (spiral motion) applying the actual values of the truncated cone structure measured by electron microscopy gave the theoretical values nearly coincident with the actual measurements. The computer animation simulated the process of the movement of the coiled filaments composing the truncated cone and suggested that the elongation of the truncated cone into thin cylinder can be elucidated as a helical movement of the coiled filaments keeping their length constant.

Quick‐freeze, deep‐etch electron microscopy further revealed that each of the coiled filaments was characterized by its beaded configuration, closely resembling that of the intermediate filametns of our previous results by immunoelectron microscopy and immunoblot analysis. The movement of the helically coiled filaments of the truncated cone may provide first example of the intermediate filaments to participate in motility and fertilization. © 1993 Wiley‐Liss, Inc.
Multiple sites for subtilisin cleavage of tubulin: Effects of divalent cations
Authors: Lobert Sharon , Hennington Bettye Sue , Correia John J.
Published: February 2005

Limited digestion of pig brain GDP‐tubulin by subtilisin was carried out in the presence of Mg2+, Mn2+, Ca2+, Zn2+, or Be2+. Isoelectric focusing, followed by SDS‐PAGE, revealed characteristic divalent cation‐dependent changes in the α‐ and β‐tubulin cleavage patterns. Previous studies revealed that the β‐cleavage pattern is different for heterodimers and microtubules [Lobert and Correia, 1992: Arch. Biochem. Biophys. 296:152–160]. Divalent cation effects on subtilisin digestion of tubulin indicate different classes of divalent cation binding sites. Western blot analysis locates the proteolytic zone at residue 430 or higher in both subunits for all conditions. Turbidity and electron microscopy reveal that GDP‐tubulin cleaved by subtilisin in the presence of Mg2+, Ca2+, or Mn2+ forms sheets of rings. Mn2+ induces ring formation in uncleaved GDP‐tubulin. Isotype‐depleted tubulin was generated by the removal of class III β‐tubulin using immunoaffinity chromatography. Subtilisin digestion of the depleted fraction and the purified class III β‐tubulin demonstrates that cleavage occurs at three to four distinct sites. Thus, subtilisin‐digested tubulin is more heterogeneous than was previously reported and the cleavage sites depend on solution conditions, divalent cations, and the state of assembly. This has important implications for experiments that utilize subtilisin‐digested tubulin for studying microtubule‐associated protein binding. © 1993 Wiley‐Liss, Inc.
Recent quantitative studies of actin filament turnover during cell locomotion
Authors: Zigmond Sally H.
Published: February 2005

Cell locomotion depends on polymerization and depolymerization of filamentous actin. Net polymerization at the cell front occurs fast enough to fill the extending lamellipod, and since total F‐actin is essentially constant over time, depolymerization must equal polymerization. Indeed, the fastest moving cell types have the highest rates of depolymerization. Accounting for the high rate of depolymerization raises several problems. One is that net depolymerization requires the concentration of G‐actin to be low (below the critical concentration), but rapid polymerization (occurring <1 μm away) requires the concentration of G‐actin to be high (well above the critical concentration). This may be accomplished by spatial compartmentalization of factors that favor polymerization or depolymerization, and/or by proteins that bind G‐actin and prevent spontaneous polymerization while allowing barbed‐end elongation. A second problem is that depolymerization proceeds faster than would seem possible from studies of F‐actin in vitro (as calculated from number and lenghts of filaments present and in vitro rate constants). Rapid depolymerization may be accomplished by filament cutters or by cytoplasmic components (as yet undiscovered) that increase the rate of depolymerization. © 1993 Wiley‐Liss, Inc.