bluuebunny/crossref_metadata_2025 huggingface dataset:
Endocytosis by human platelets: metabolic and freeze-fracture studies.
Authors: Zucker-Franklin D
Published: May 2004

The mechanism by which platelets endocytose or release particulate or soluble substances is poorly understood. Engulfed materials enter the open canalicular system (OCS) by a process akin to phagocytosis, but fusion of platelet granules with the OCS is rarely observed. Secretion of granule contents, a concomitant of the "release reaction" which occurs during platelet aggregation, does not take place by extrusion at the surface membrane as is true for other secretory cells. Some substances may be secreted without obvious granule loss. To examine whether structural properties of the platelet membrane could account for this unusual behavior, thin section and freeze-fracture analyses were performed on platelets which had undergone endocytosis under a variety of experimental conditions. After freeze-cleavage, most of the intramembranous particles (IMP) remain associated with the outer leaflet of the platelet plasma membrane. The sites where the OCS reaches the surface membrane are marked by pits on the cytoplasmic leaflet (P face) and by complementary protrusions on the outer leaflet (E face) of the membrane. Endocytosis of small particles and solutes takes place via these structures. This process is not energy dependent but arrested at 4 degrees C. Distension of the OCS does not appear to affect the size or number of the pits. On the other hand, large particles are taken up by membrane invagination without redistribution of IMP's and independent of the pits. This process is sensitive to metabolic inhibition. Thus, the studies have demonstrated the existence of two different pathways for platelet endocytosis which are postulated to be also involved in secretion. The selective release of substances contained in different granules may be related to the "inside-out" structure of the plasma and OCS membranes.
Mechanism of formation, ultrastructure, and function of the cytoplasmic bridge system during morphogenesis in Volvox.
Authors: Green K J , Viamontes G I , Kirk D L
Published: May 2004

The cytoplasmic bridge system that links all cells of a Volvox embryo and plays a crucial role in morphogenesis is shown to form as a result of localized incomplete cytokinesis; sometimes bridge formation occurs before other regions of the cell have begun to divide. Vesicles, believed to be derived from the cell interior, align along the presumptive cleavage furrow in the bridge-forming region. Apparently it is where these vesicles fail to fuse that bridges are formed. Conventional and high voltage transmission electron microscopy analyses confirm that bridges are regularly spaced; they possess a constant, highly ordered structure throughout cleavage and inversion. Concentric cortical striations (similar to those observed previously in related species) ring each bridge throughout its length and continue out under the plasmalemma of the cell body to abut the striations of neighboring bridges. These striations are closely associated with an electron-dense material that coats the inner face of the membrane throughout the bridge region and appears to be thickest near the equator of each bridge. In addition to the parallel longitudinal arrays of cortical microtubules that traverse the cells, we observed microtubules that angle into and through the bridges during cleavage; however, the latter are not seen once inversion movements have begun. During inversion, bridge bands undergo relocation relative to the cell bodies without any loss of integrity or change in bridge spacing. Observation of isolated cell clusters reveals that it is the sequential movement of individual cells with respect to a stationary bridge system, and not actual movement of the bridges, that gives rise to the observed relocation.
Centriole cycle in Chinese hamster ovary cells as determined by whole-mount electron microscopy.
Authors: Kuriyama R , Borisy G G
Published: May 2004

In interphase Chinese hamster ovary (CHO) cells, the centrosome is attached to the nucleus very firmly. This nuclear-centrosome complex is isolated as a coherent structure by lysis and extraction of cells with Triton X-100 in a low ionic strength medium. Under these conditions, the ultrastructure of the centrioles attached to the nucleus can be discerned by electron microscopy of whole-mount preparations. The structural changes of the centrioles as a function of the cell cycle were monitored by this technique. Specifically, centriolar profiles were placed into six categories according to their orientation and the length ratio of daughter and parent centrioles. The proportion of centrioles in each category was plotted as a frequency histogram. The morphological changes in the centriole cycle were characterized by three distinguishable events: nucleation, elongation, and disorientation. The progress of centrioles through these stages was determined in synchronous populations of cells starting from S or M phase, in cells inhibited in DNA synthesis by addition of thymidine, and in cytoplasts. The results provide a quantitative description of the events of the centriole cycle. They also show that, in complete cells, nucleation, elongation, and disorientation are not dependent upon DNA synthesis. However, in cytoplasts, although elongation and disorientation occur as in normal cells, nucleation is blocked. Procentriole formation appeared to be inhibited by the removal of the nucleus. We suggest that coordination of centriole replication and nuclear replication may depend upon a signal arising from the nucleus.
Microtubule-nucleating activity of centrosomes in Chinese hamster ovary cells is independent of the centriole cycle but coupled to the mitotic cycle.
Authors: Kuriyama R , Borisy G G
Published: May 2004

The nuclear-centrosome complex was isolated from interphase Chinese hamster ovary (CHO) cells, and, with exogenous brain tubulin as a source of subunits, the centrosome, while attached to the nucleus, was demonstrated to nucleate microtubule formation in vitro. We attempted to quantitate the nucleating activity in order to compare the activity of mitotic and interphase centrosomes. However, the proximity of the nucleus hindered these attempts, and efforts to chemically or mechanically remove the centrosome led to diminished nucleating activity. Therefore, the nuclear-centrosome complex was dissociated biologically through use of the cytochalasin B procedure for enucleation of cells. Cytoplasts were prepared that retained the centrosome. Lysis of the cytoplasts released free centrosomes that could nucleate microtubules in vitro. The nucleating activities of interphase and mitotic centrosomes were compared. In addition, through the use of whole-mount electron microscopy, the configuration of the centrioles was analyzed and the number of microtubules nucleated was determined as a function of the centriole cycle. Nucleating activity did not change discernibly throughout interphase but increased approximately fivefold at the transition to mitosis. Thus, we conclude that the nucleating activity of the centrosome is relatively independent of the centriole cycle but coupled to the mitotic cycle.
Characterization of a myoepithelial cell line derived from a neonatal rat mammary gland.
Authors: Warburton M J , Ormerod E J , Monaghan P , Ferns S , Rudland P S
Published: May 2004

A clonal, myoepithelial-like cell line has been obtained from a primary culture established from the mammary gland of a 7-d-old rat. In a number of respects, this cell line, termed Rama 401, resembles the myoepithelial cells of the mammary gland, especially when grown on floating collagen gels. The cells grow as multilayers on the gel surface and form branching structures that do not appear to contain a lumen. They are rather elongated, with irregular-shaped, flattened nuclei that contain large amounts of peripheral chromatin. Elongated processes project from the cell surface and numerous membrane pinocytotic vesicles can be seen. The cytoplasm is filled with linear arrays of 5- to 7-nm filaments with occasional dense foci. Cell junctions with associated 8- to 11-nm tonofilaments are also observed. Immunofluorescence techniques reveal actin and myosin filaments and also intermediate filaments of both prekeratin and vimentin types. Rama 401 cells secrete an amorphous material that, when an immunoperoxidase technique is used, stains with antibodies to basement membrane-specific type IV collagen. Localized densities of the cell membrane, which resemble hemidesmosomes, are located adjacent to these extracellular deposits. Immunofluorescence staining and immunoprecipitation techniques reveal that the cells also synthesize two other basement membrane proteins, laminin and fibronectin. The type IV collagen consists of two chains with molecular weights of 195,000 and 185,000.
Control of the erythrocyte membrane shape: recovery from the effect of crenating agents.
Authors: Alhanaty E , Sheetz M P
Published: May 2004

Intact erythrocytes become immediately crenated upon addition of 2,4-dinitrophenol (DNP) or pyrenebutyric acid (PBA). However, when cells are incubated at 37 degrees C in the presence of the crenating agents with glucose, they gradually (4--8 h) recover the normal biconcave disc form. The recovery process does not reflect a gradual inactivation of DNP or PBA since fresh cells are equally crenated by the supernatant from the recovered cells. Further, after recovery and removal of the crenating agents, cells are found to be desensitized to the readdition of DNP as well as to the addition of PBA, but they are more sensitive to cupping by chlorpromazine. This alteration in the cell membrane responsiveness was reversible upon further incubation in the absence of DNP. Recovery is dependent upon cellular metabolic state since an energy source is needed and incubation with guanosine but not adenosine will accelerate conversion to the disc shape. It is suggested that the conversion of cells from crenated to disc shape in the presence of the crenators, represents an alteration or rearrangement of membrane components rather than a redistribution of the crenators within the membrane. This shape recovery process may be important for erythrocyte shape preservation as well as shape control in other cells.
Phosphorylation of myosin light chain during capping of mouse T-lymphoma cells.
Authors: Bourguignon L Y , Nagpal M L , Hsing Y C
Published: May 2004

Colchicine induces the clustering of at least three different T-lymphoma surface antigens (T200, Thy-1, and gp 69/71) into a cap structure in the absence of any external ligand. In addition, colchicine induces the intracellular accumulation of actin and myosin directly beneath the surface cap structure. We have discovered that myosin molecules (both heavy and light chains) are closely associated with the plasma membrane of T-lymphoma cells. Most importantly, we have found that the 20,000-dalton light chain of lymphocyte myosin is both phosphorylated and preferentially accumulated in the plasma membrane of colchicine-induced capped cells. It is proposed that myosin light chain is directly involved in the activation of membrane-associated actomyosin required for the collection of surface proteins into a cap structure (analogous to muscle cell sliding filament contraction).
Intercellular junctions and transfer of small molecules in primary vascular endothelial cultures.
Authors: Larson D M , Sheridan J D
Published: May 2004

The ultrastructure of gap and tight junctions and the cell-to-cell transfer of small molecules were studied in primary cultures and freshly isolated sheets of endothelial cells from calf aortae and umbilical veins. In thin sections and in freeze-fracture replicas, the gap and tight junctions in the freshly isolated cells from both sources appeared similar to those found in the intimal endothelium. Most of the interfaces in replicas had complex arrays of multiple gap junctions either intercalated within tight junction networks or interconnected by linear particle strands. The particle density in the center of most gap junctions was noticeably reduced. In confluent monolayers, after 3-5 days in culture, gap and tight junctions were present, although reduced in complexity and apparent extent. Despite the relative simplicity of the junctions, the cell-to-cell transfer of potential changes, dye (Lucifer Yellow CH), and nucleotides was readily detectable in cultures of both endothelial cell types. The extent and rapidity of dye transfer in culture was only slightly less than that in sheets of freshly isolated cells, perhaps reflecting a reduced gap junctional area combined with an increase in cell size in vitro.
Reactivation of contraction in detergent-lysed teleost retinal cones.
Authors: Burnside B , Smith B , Nagata M , Porrello K
Published: May 2004

Teleost retinal cones contract in the light and elongate in the dark. In the green sunfish, Lepomis cyanellus, the necklike myoid region of the cone contracts from as much as 120 micrometers (midnight dark-adapted) to 6 micrometers in fully light-adapted state. When dark-adapted fish are exposed to light (1.4 lux), cone myoids contract with a linear rate of 1.5 +/- 0.1 micrometers/min. We report here that detergent-lysed motile models of teleost retinal cones exhibit calcium- and ATP-dependent reactivated contraction, with morphology and rate comparable to that observed in vivo. For reactivation studies isolated dark-adapted retinas were lysed with nonionic detergent Brij-58 (0.1-1.0%). In reactivation medium containing 10(-5) M free calcium and 4 mM ATP, the lysed cones contracted with normal morphology at in vivo rates (1.4 +/- 1 micrometer/min). Little contraction was observed if ATP or detergent was deleted from the medium or if free calcium levels were less than 10(-8) M. Ultrastructural examination of cone models lysed with 1% Brij-58 revealed that, in spite of extensive extraction of the cytoplasmic matrix, cytoskeletal components (thin filaments, intermediate filaments, microtubules) were still present. Thus we have produced extensively extracted motile models of teleost retinal cones which undergo calcium- and ATP-dependent reactivated contraction with normal morphology at physiological rate.
Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the M(r) 43,000 nonreceptor peptide
Authors: Barrantes FJ
Published: May 2004

Oligomeric forms of the acetylcholine receptor are directly visualized by electron microscopy in receptor-rich membranes from torpedo marmorata. The receptor structures are quantitatively correlated with the molecular species so far identified only after detergent solubilization, and further related to the polypeptide composition of the membranes and changes thereof. The structural identification is made possibly by the increased fragility of the membranes after extraction of nonreceptor peptides and their subsequent disruption upon drying onto hydrophilic carbon supports.

Receptor particles in native membranes depleted of nonreceptor peptides appear as single units of 7-8 nm, and double and multiple aggregates thereof. Particle doublets having a main-axis diameter of 19 +/- 3 nm predominate in these membranes. Linear aggregates of particles similar to those observed in rotary replicas of quick-frozen fresh electrolytes (Heuser, J.E. and S. R. Salpeter. 1979, J. Cell Biol. 82: 150-173) are also present in the alkaline-extracted membranes. Chemical modifications of the thiol groups shift the distribution of structural species. Dithiothreitol reduction, which renders almost exclusively the 9S, monomeric receptor form, results in the observation of the 7-8 nm particle in isolated form. The proportion of doublets increases in membranes alkylated with N-ethylmaleimide. Treatment with 5,5'-dithiobis-(nitrobenzoic acid) increases the proportion of higher oligomeric species, and particle aggregates (n=oligo) predominate.

The nonreceptor v-peptide (doublet of M(r) 43,000) appears to play a role in the receptor monomer-polymer equilibria. Receptor protein and v-peptide co-aggregate upon reduction and reoxidation of native membranes. In membranes protected ab initio with N- ethylmaleimide, only the receptor appears to self-aggregate. The v-peptide cannot be extracted from these alkylated membranes, though it is easily released from normal, subsequently alkylated or reduced membranes. A stabilization of the dimeric species by the nonreceptor v-peptide is suggested by these experiments.

Monospecific antibodies against the v-peptide are used in conjunction with rhodamine- labeled anti-bodies in an indirect immunoflourescence assay to map the vectorial exposure of the v-peptide. When intact membranes, v-peptide depleted and "holey" native membranes (treated with 0.3 percent saponin) are compared, maximal labeling is obtained with the latter type of membranes, suggesting a predominantly cytoplasmic exposure of the antigenic determinants of the v-peptide in the membrane.

The influence of the v-peptide in the thiol-dependent interconversions of the receptor protein and the putative topography of the peptide are analyzed in the light of the present results.
In situ reconstitution of myosin filaments within the myosin-extracted myofibril in cultured skeletal muscle cells.
Authors: Taniguchi M , Ishikawa H
Published: May 2004

We studied the in situ reconstitution of myosin filaments within the myosin-extracted myofibrils in cultured chick embryo skeletal muscle cells using the electron microscope and polarization microscope. Myosin was first extracted from the myofibrils in glycerinated muscle cells with a high-salt solution containing 0.6 M KCl. When rabbit skeletal muscle myosin was added to the myosin-extracted cells in the high-salt solution, thin filaments in the ghost myofibrils were bound with myosin to form arrowhead complexes. Subsequent dilution of KCl in the myosin solution to 0.1 M resulted in the formation of thick myosin filaments within the myofibrils, increasing the birefringence of the myofibrils. When Mg-ATP was added such myosin-reassembled myofibrils were induced either to form supercontraction bands or to restore the sarcomeric arrangement of thick and thin filaments. Under the polarization microscope, vibrational movement of the myofibrils was seen transiently upon addition of Mg-ATP, often resulting in a regular arrangement of myofibrils in register. These myofibrils, with reconstituted myosin filaments, structurally and functionally resembled the native myofibrils. The findings are discussed with special reference to the myofibril formation in developing muscle cells.
Ultrastructural localization of the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum in rat skeletal muscle by immunoferritin labeling of ultrathin frozen sections.
Authors: Jorgensen A O , Shen A C , MacLennan D H , Tokuyasu K T
Published: May 2004

The ultrastructural localization of the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum in rat gracilis muscle was determined by indirect immunoferritin labeling of ultrathin frozen sections. Simultaneous visualization of ferritin particles and of adsorption-stained cellular membranes showed that the Ca2+ + Mg2+-ATPase was concentrated in the longitudinal sarcoplasmic reticulum and in the nonjunctional regions of the terminal cisternae membrane but was virtually absent from mitochondria, plasma membranes, transverse tubules, and junctional sarcoplasmic reticulum. Ferritin particles were found preponderantly on the cytoplasmic surface of the membrane, in agreement with published data showing an asymmetry of the Ca2+ + Mg2+-ATPase within the sarcoplasmic reticulum membrane. Comparison of the density of ferritin particles in fast and slow myofibers suggested that the density of the Ca2+ + Mg2+-ATPase in the sarcoplasmic reticulum membrane in a fast myofiber is approximately two times higher than in a slow myofiber.
Microtubules and beta cell function: effect of colchicine on microtubules and insulin secretion in vitro by mouse beta cells.
Authors: Boyd A E , Bolton W E , Brinkley B R
Published: May 2004

A monolayer culture system was developed to study the role of microtubules in insulin secretion. Cultured cells were obtained to study the role of microtubules in insulin secretion. Cultured cells were obtained by enzymatic digestion of pancreases from C57BL-KsJ mice 6-12 wk of age. On day 4 of culture, the medium was changed, control or treatment medium added, and frequent samples were removed for insulin assay. Microtubules and beta cells were identified by indirect immunofluorescence with monospecific antibodies to tubulin and insulin. An extensive microtubule network radiates from the perinuclear region of the beta cell to the plasma membrane. Although alterations in the calcium concentration of the medium did not affect the microtubule pattern, the absence of calcium or glucose in the medium inhibited insulin secretion (P less than 0.001). Optimum insulin release occurred at a calcium concentration of 2.5 mM. Colchicine, in concentrations of 10(-10) M, did not affect the microtubule immunofluorescent pattern, whereas concentrations of 1 and 5 x 10(-7) M decreased the number of microtubules, and microtubules could not be identified in cultures treated with 10(-6) M colchicine for 2 h. After a 2-h preincubation, the prolonged release of insulin at either 2.0 or 4.5 mg/ml of glucose was decreased by 10(-6) M colchicine (P less than 0.02). The immediate release of insulin was similar to that in control plates and occurred in cultures with no identifiable microtubules. Microtubules and insulin secretion were not altered by 10(-6) M lumicolchicine and prolonged insulin secretion recovered 24 h after removal of colchicine. These studies show that the microtubules facilitate sustained secretion of insulin but are not required for the immediate release of the hormone. Alterations in the extracellular calcium concentration which play an essential role in insulin secretion do not alter the microtubule pattern in the beta cell.
A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs).
Authors: Vallee R B
Published: May 2004

The effect of the antimitotic drug taxol on the association of MAPs (microtubule-associated proteins) with microtubules was investigated. Extensive microtubule assembly occurred in the presence of Taxol at 37 degrees C. at 0 degrees C, and at 37 degrees C in the presence of 0.35 M NaCl, overcoming the inhibition of assembly normally observed under the latter two conditions. At 37 degrees C and at 0 degrees C, complete assembly of both tubulin and the MAPs was observed in the presence of Taxol. However, at elevated ionic strength, only tubulin assembled, forming microtubules devoid of MAPs. The MAPs could also be released from the surface of preformed microtubules by exposure to elevated ionic strength. These properties provided the basis for a rapid new procedure for isolating microtubules and MAPs of high purity from small amounts of biological material. The MAPs could be recovered by exposure of the microtubules to elevated ionic strength and subjected to further analysis. Microtubules and MAPs were prepared from bovine cerebral cortex (gray matter) and from HeLa cells. MAP 1, MAP2, and the tau MAPs, as well as species of Mr = 28,000 and 30,000 (LMW, or low molecular weight, MAPs) and a species of Mr = 70,000 were isolated from gray matter. Species identified as the 210,000 and 125,000 mol wt HeLa MAPs were isolated from HeLa cells. Microtubules were also prepared for the first time from white matter. All of the MAPs identified in gray matter preparations were identified in white matter, but the amounts of individual MAP species differed. The most striking difference in the two preparations was a fivefold lower level of MAP 2 relative to tubulin in white matter than in gray. The high molecular weigh MAP, MAP1, was present in equal ratio to tubulin in white and gray matter. These results indicate that MAP 1 and MAP2, as well as other MAP species, may have a different cellular or subcellular distribution.
An electron microscopic and optical diffraction analysis of the structure of Limulus telson muscle thick filaments.
Authors: Kensler R W , Levine R J
Published: May 2004

Long, thick filaments (greater than 4.0 micrometer) rapidly and gently isolated from fresh, unstimulated Limulus muscle by an improved procedure have been examined by electron microscopy and optical diffraction. Images of negatively stained filaments appear highly periodic with a well-preserved myosin cross-bridge array. Optical diffraction patterns of the electron micrographs show a wealth of detail and are consistent with a myosin helical repeat of 43.8 nm, similar to that observed by x-ray diffraction. Analysis of the optical diffraction patterns, in conjunction with the appearance in electron micrographs of the filaments, supports a model for the filament in which the myosin cross-bridges are arranged on a four-stranded helix, with 12 cross-bridges per turn or each helix, thus giving an axial repeat every third level of cross-bridges (43.8 nm).
Changes in the components of extracellular matrix and in growth properties of cultured aortic smooth muscle cells upon ascorbate feeding.
Authors: Schwartz E , Bienkowski R S , Coltoff-Schiller B , Goldfischer S , Blumenfeld O O
Published: May 2004

Culture conditions can modify the composition of the extracellular matrix of cultured calf aortas smooth muscle cells. In the absence of ascorbate the major components of the matrix are microfibrillar proteins; deposition of collagen occurs upon ascorbate supplementation and, with increased time of exposure of cells to ascorbate, collagen becomes the dominant protein of the extracellular matrix (greater than 80%). Collagen accumulation follows a sigmoidal time-course, suggesting that it is a cooperative phenomenon. Covalent crosslinks are not required for collagen accumulation in the matrix. Microfibrillar proteins and increased amounts of proteoglycans and fibronectin accumulate concurrently with collagen but elastin deposition was not observed either with or without ascorbate feeding. Addition of ascorbate leads to a general stimulation of incorporation of [14C]proline into cellular protein and to changes in cell growth parameters and morphology: cell-doubling time decreases from 62 to 47 h and plating efficiency increases approximately fourfold. We conclude that the composition of the extracellular matrix assembled by cultured cells is subject to experimental manipulation and that changes in endogenously deposited matrix may have significant effects on cellular functions.
Dynamics of spindle microtubule organization: kinetochore fiber microtubules of plant endosperm.
Authors: Jensen C G
Published: May 2004

Organization of kinetochore fiber microtubules (MTs) throughout mitosis in the endosperm of Haemanthus katherinae Bak. has been analysed using serial section reconstruction from electron micrographs. Accurate and complete studies have required careful analysis of individual MTs in precisely oriented serial sections through many (45) preselected cells. Kinetochore MTs (kMTs) and non-kinetochore MTs (nkMTs) intermingle within the fiber throughout division, undergoing characteristic, time-dependent, organizational changes. The number of kMTs increases progressively throughout the kinetochore during prometaphase-metaphase. Prometaphase chromosomes which were probably moving toward the pole at the time of fixation have unequally developed kinetochores associated with many nkMTs. The greatest numbers of kMTs (74-109/kinetochore), kinetochore cross-sectional area, and kMT central density all occur at metaphase. Throughout anaphase and telophase there is a decrease in the number of kMTs and, in the kinetochore cross-sectional area, an increased obliquity of kMTs and increased numbers of short MTs near the kinetochore. Delayed kinetochores possess more kMTs than do kinetochores near the poles, but fewer kMTs than chromosomes which have moved equivalent distances in other cells. The frequency of C-shaped proximal MT terminations within kinetochores is highest at early prometaphase and midtelophase, falling to zero at midanaphase. Therefore, in Haemanthus, MTs are probably lost from the periphery of the kinetochore during anaphase in a manner which is related to both time and position of the chromosome along the spindle axis. The complex, time-dependent organization of MTs in the kinetochore region strongly suggests that chromosome movement is accompanied by continual MT rearrangement and/or assembly/disassembly.
Microinjection of cytoplasm as a test of complementation in Paramecium.
Authors: Haga N , Forte M , Saimi Y , Kung C
Published: May 2004

Mutants in Paramecium tetraurelia, unable to generate action potentials, have been isolated as cells which show no backward swimming in response to ionic stimulation. These "pawn" mutants belong to at least three complementation groups designated pwA, pwB, and pwC. We have found that microinjection of cytoplasm from a wild-type donor into a pawn recipient of any of the three complementation groups restores the ability of the pawn to generate action potentials and hence swim backward. In addition, the cytoplasm from a pawn cannot restore a recipient of the same complementation group, but that from a pawn of a different group can. Electrophysiological analysis had demonstrated that the restoration of backward swimming is not due to a simple addition of ions but represents a profound change in the excitable membrane of the recipient pawn cells. Using known pawn mutants and those which had previously been unclassified, we have been able to establish a perfect concordance of genetic complementation and complementation by cytoplasmic transfer through microinjection. This method has been used to classify pawn mutants that are sterile or hard-to-mate and to examine the ability of cytoplasms from different species of ciliated protozoa to restore the ability to swim backward in the pawn mutants of P. tetraurelia. A cell homogenate has also been fractionated by centrifugation to further purify the active components. These results demonstrate that transfer of cytoplasm between cells by microinjection can be a valid and systematic method to classify mutants. This test is simpler to perform than the genetic complementation test and can be used under favorable conditions in mutants that are sterile and in cells of different species.
Assembly of keratin onto PtK1 cytoskeletons: evidence for an intermediate filament organizing center.
Authors: Eckert B S , Daley R A , Parysek L M
Published: May 2004

Purified keratin, solubilized in 8 M of urea, was added to Triton X-100-extracted PtK1 cells in 5 mM PIPES buffer. The buffer conditions induced assembly of keratin filaments which appear to associate with nuclei of extracted cells. These keratin fibers extend beyond the original margin of the cells and frequently form bridges between adjacent cells. Electron microscopy shows that keratin filaments associate closely with the surface of the nucleus. We suggest that the site of association between keratin and the nucleus may represent an intermediate filament organizing center.
Defective internalization of low density lipoprotein in epidermoid cervical cancer cells.
Authors: Gal D , Simpson E R , Porter J C , Snyder J M
Published: May 2004

Cells of an epidermoid cancer cell line of human uterine cervix, which possessed a high-affinity, specific receptor for low density lipoprotein (LDL), internalized and degraded [125I]iodo-LDL at a very low rate. In these cells, LDL did not stimulate cholesteryl ester synthesis, nor did it suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase to the same extent as in the control cells. The binding of [125I]iodo-LDL by these cells was not decreased by preincubation of the cells in medium containing LDL. Using ferritin-labeled LDL (F-LDL) and electron microscopy, it was determined that at 4 degrees C the cells bound F-LDL in the same way as other cancer cell lines that did not have a defect in internalization. When these cells were warmed to 37 degrees C the F-LDL remained on the surface, whereas in cells from control cancer cell lines the F-LDL was internalized and was no longer observed on the cell surface. On the basis of the results of these studies it is concluded that cells of this epidermoid cancer cell line have a defective ability to internalize LDL.
Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization.
Authors: Jaffe L A , Gould-Somero M , Holland L Z
Published: May 2004

Prevention of polyspermic fertilization in sea urchins (Jaffe, 1976, Nature (Lond.). 261:68-71) and the worm Urechis (Gould-Somero, Jaffe, and Holland, 1979, J. Cell Biol. 82:426-440) involves an electrically mediated fast block. The fertilizing sperm causes a positive shift in the egg's membrane potential; this fertilization potential prevents additional sperm entries. Since in Urechis the egg membrane potential required to prevent fertilization is more positive than in the sea urchin, we tested whether in a cross-species fertilization the blocking voltage is determined by the species of the egg or by the species of the sperm. With some sea urchin (Strongylocentrotus purpuratus) females, greater than or equal to 90% of the eggs were fertilized by Urechis sperm; a fertilization potential occurred, the fertilization envelope elevated, and sometimes decondensing Urechis sperm nuclei were found in the egg cytoplasm. After insemination of sea urchin eggs with Urechis sperm during voltage clamp at +50 mV, fertilization (fertilization envelope elevation) occurred in only nine of twenty trials, whereas, at +20 mV, fertilization occurred in ten of ten trials. With the same concentration of sea urchin sperm, fertilization of sea urchin eggs occurred, in only two of ten trials at +20 mV. These results indicate that the blocking voltage for fertilization in these crosses is determined by the sperm species, consistent with the hypothesis that the fertilization potential may block the translocation within the egg membrane of a positively charged component of the sperm.
Characterization of a cell cycle mutant derived from hamster fibroblast: reversion analysis.
Authors: Scharff D J , Delegeane A M , Lee A S
Published: May 2004

K12 is a temperature-sensitive (ts) mutant cell line derived from Chinese hamster fibroblasts. When incubated at the nonpermissive temperature, K12 cells exhibit the following properties: (a) the cells cannot initiate DNA synthesis;o (b) the synthesis of cytosol thymidine kinase is suppressed; and (c) the synthesis of three cellular proteins of molecular weights 94, 78, and 58 kdaltons is greatly enhanced. Here we characterize a spontaneous revertant clone, R12, derived from the K12 cells. We selected the revertant clone for its ability to grow at the nonpermissive temperature. Our results indicate that all the traits which constitute the K12 mutant phenotype are simultaneously reverted to the wild type in the revertant cell line, suggesting that the ts mutation of the K12 cells is of regulatory nature and exerts multiple effects on the expressed phenotypes.
Partial reconstruction of the microvillus core bundle: characterization of villin as a Ca(++)-dependent, actin-bundling/depolymerizing protein
Authors: Matsudaira PT , Burgess DR
Published: May 2004

The brush border, isolated from chicken intestine epithelial cells, contains the 95,000 relative molecular mass (M(r)) polypeptide, villin. This report describes the purification and characterization of villin as a Ca(++)-dependent, actin bundling/depolymerizing protein. Then 100,000 g supernatant from a Ca(++) extract of isolated brush borders is composed of three polypeptides of 95,000 (villin), 68,000 (fimbrin), and 42,000 M(r) (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. Electron microscopy of negatively stained preparations of these villin-actin mixtures showed that filament bundles were present. This viscosity, sedimentability, and ultrastructural morphology of filament bundles are dependent on the villin:actin molar ratio, the pH, and the free Ca(++) concentration in solution. At low free Ca(++) (less than 10(-6) M), the amount of protein in bundles, when measured by sedimentation, increased as the villin: actin molar ratio increased and reached a plateau at approximately a 4:10 ratio. This behavior correlates with the conversion of single actin filaments into filament bundles as detected in the electron microscope. At high free Ca(++) (more than 10(-6) M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer. Furthermore, these Ca(++) effects were correlated with the loss of protein sedimented, the disappearance of filament bundles, and the appearance of short fragments of filaments. Bundle formation is also pH-sensitive, being favored at mildly acidic pH. A decrease in the pH from 7.6 to 6.6 results in an increase in sedimentable protein and also a transformation of loosly associated actin filaments into compact actin bundles. These results are consistent with the suggestions that villin is a bundling protein in the microvillus and is responsible for the Ca(++)-sensitive disassembly of the microvillar cytoskeleton. Thus villin may function in the cytoplasm as a major cytoskeletal element regulating microvillar shape.
Dye transfer between cells of the embryonic chick lens becomes less sensitive to CO2 treatment with development.
Authors: Schuetze S M , Goodenough D A
Published: May 2004

During the 3-h developmental stage 14 in the chick, intercellular transfer of iontophoresed fluorescent dyes becomes less sensitive to the lowering of intracellular pH by either CO2 or acetate ions. Up to developmental state 14, dye transfer between lens cells is reversibly blocked by exposure to 50% CO2. Beyond stage 14, dye transfer between these cells is no longer reversibly blocked by elevated pCO2. Electronic coupling is present throughout lens development and is not reversibly blocked by high pCO2 at any stage. The gap junctions joining the lens cells show morphological changes at developmental stage 14. Up to stage 14, all gap junctions observed between chick lens cells have connexon assemblies that appear condensed or crystalline following routine freeze-fracture microscopy. Beyond stage 14, chick lens cells express gap junctions with both the condensed assemblies and the dispersed assemblies characteristic of adult lens gap-junction structure.
Lymphocyte locomotion and attachment on two-dimensional surfaces and in three-dimensional matrices
Authors: Haston WS , Shields JM , Wilkinson PC
Published: May 2004

The adhesion and locomotion of mouse peripheral lymph node lymphocytes on 2-D protein- coated substrata and in 3-D matrices were compared. Lymphocytes did not adhere to, or migrate on, 2-D substrata suck as serum- or fibronectin-coated glass. They did attach to and migrate in hydrated 3-D collagen lattices. When the collagen was dehydrated to form a 2-D surface, lymphocyte attachment to it was reduced. We propose that lymphocytes, which are poorly adhesive, are able to attach to and migrate in 3-D matrices by a nonadhesive mechanism such as the extension and expansion of pseudopodia through gaps in the matrix, which could provide purchase for movement in the absence of discrete intermolecular adhesions. This was supported by studies using serum-coated micropore filters, since lymphocytes attached to and migrated into filters with pore sizes large enough (3 or 8 mum) to allow pseudopod penetration but did not attach to filters made of an identical material (cellulose esters) but of narrow pore size (0.22 or 0.45 mum). Cinematographic studies of lymphocyte locomotion in collagen gels were also consistent with the above hypothesis, since lymphocytes showed a more variable morphology than is typically seen on plane surfaces, with formation of many small pseudopodia expanded to give a marked constriction between the cell and the pseudopod. These extensions often remained fixed with respect to the environment as the lymphocyte moved away from or past them. This suggests that the pseudopodia were inserted into gaps in the gel matrix and acted as anchorage points for locomotion.
Immunocytochemical localization of wheat germ agglutinin in wheat.
Authors: Mishkind M , Raikhel N V , Palevitz B A , Keegstra K
Published: May 2004

Immunocytological techniques were developed to localize the plant lectin, wheat germ agglutinin (WGA), in the tissues and cells of wheat plants. In a previous study we demonstrated with a radioimmunoassay that the lectin is present in wheat embryos and adult plants both in the roots and at the base of the stem. We have now found, using rhodamine, peroxidase, and ferritin-labeled secondary antibodies, that WGA is located in cells and tissues that establish direct contact with the soil during germination and growth of the plant In the embryo, WGA is found in the surface layer of the radicle, the first adventitious roots, the coleoptile, and the scutellum. Although found throughout the coleorhiza and epiblast, it is at its highest levels within the cells at the surface of these organs. In adult plants, WGA is located only in the caps and tips of adventitious roots. Reaction product for WGA was not visualized in embryonic or adult leaves or in other tissues of adult plants. At the subcellular level, WGA is located at the periphery of protein bodies, within electron-translucent regions of the cytoplasm, and at the cell wall-protoplast interface. Since WGA is found at potential infection sites and is known to have fungicidal properties, it may function in the defense against fungal pathogens.
Elimination of specific DNA sequences from the somatic nucleus of the ciliate Tetrahymena.
Authors: Yao M C
Published: May 2004

Tetrahymena micronuclear DNA fragments have been cloned in the plasmid pBR322. One clone, pTt 2512, has been found to contain the C-C-C-C-A-A hexanucleotide repeat which is also present in the macronuclear rDNA. Further restriction enzyme digestion and hybridization studies suggest that the clone also contains sequences that are not present in the somatic macronucleus. The flanking sequences of the C4A2 repeats in this clone were separated into four restriction fragments, one from one side and three from the other. These fragments were used as probes for Southern hybridization to study the organizations of similar sequences in the macronucleus and micronucleus. All four fragments hybridized to many fragments of restriction enzyme digested micronuclear DNA. However, none of these hybridizations were detected in the macronucleus. Thus, these families of repetitive DNA are completely eliminated from the macronucleus. Further analysis suggested that the four different sequences may be linked at other locations of the genome. Using nullisomic strains of Tetrahymena, it is found that at least one of these sequences is present in more than one chromosome. Studies of various normal and star strains of Tetrahymena suggest that these sequences are stable in the normal micronucleus but are altered drastically in the defective micronuclei of the star strains. Eliminated DNA of similar nature has also been found in at least five other randomly selected clones of micronuclear DNA and may be present widely in the genome.
Cell behavior in Dictyostelium discoideum: preaggregation response to localized cyclic AMP pulses.
Authors: Futrelle R P , Traut J , McKee W G
Published: May 2004

The motion of cells in the aggregation phase of Dictyostelium discoideum development is complex. To probe its mechanisms we applied precisely timed (+/- 1 s) and positioned (+/-2 micrometers) pulses of cyclic AMP to fields of cells of moderate density using a micropipette. We recorded cell behavior by time lapse microcinematography and extracted cell motion data from the film with our Galatea computer system. Analysis of these data reveals: (a) Chemotaxis lasts only about as long as the cyclic AMP signal; in particular, brief pulses (approximately 5 s) do not induce chemotaxis. (b) Chemotactic competence increases gradually from within an hour after the initiation of development (starvation) to full competence at approximately 15 h when aggregation begins under our conditions. (c) Cell motion reverses rapidly (within 20 s) when the external gradient is reversed. There is no refractory period for motion. We present a new description of the process of aggregation consistent with our result and other recent findings. (d) The behavioral response to cyclic AMP includes a phenomenon we call "cringing." In a prototypical cringe the cell speed drops within 3 s after a brief cyclic AMP stimulus, and the cell stops and rounds and then resumes motion after 25 s. (e) The development of the speed response in cringing as the cells age closely parallels the development of the cyclic AMP-induced light-scattering response of cells in suspension. (f) Cringing occurs in natural populations during weak oriented movement. The computerized analysis of cell behavior proves to be a powerful technique which can reveal significant phenomena that are not apparent to the eye even after repeated examination of the film.
Hepatic Golgi fractions resolved into membrane and content subfractions.
Authors: Howell K E , Palade G E
Published: May 2004

Golgi fractions isolated from rat liver homogenates have been resolved into membrane and content subfractions by treatment with 100 mM Na2CO3 pH 11.3. This procedure permitted extensive extraction of content proteins and lipoproteins, presumably because it caused an alteration of Golgi membranes that minimized the reformation of closed vesicles. The type and degree of contamination of the fractions was assessed by electron microscopy and biochemical assays. The membrane subfraction retained 15% of content proteins and lipids, and these could not be removed by various washing procedures. The content subfraction was contaminated by both membrane fragments and vesicles and accounted for 5 to 10% of the membrane enzyme activities of the original Golgi fraction. The lipid compositions of the subfractions was determined, and the phospholipids of both membrane and content were found to be uniformly labeled with [33P]phosphate administered in vivo.
Histones synthesized for use in early development of Xenopus laevis are stored as a complex with antigenic properties similar to those of the octamer core of nucleosomes.
Authors: Earnshaw W C , Rekvig O P , Hannestad K
Published: May 2004

Serum of patients with systemic lupus erythematosus (SLE) contains crossreacting autoantibodies which recognize histones in nucleosomes or when they are induced to form octamers in solution in the presence of 2 M NaCl, but not when they are dissociated free in solution at physiological ionic strength. We have found that histones stored in eggs of Xenopus laevis for use in rapid nuclear synthesis during early development react with this antibody. This reaction has been observed by radioimmunoassay, inhibition of chromatin assembly by the extracts in the presence of antibody, and, in a preliminary result, by identification of a histone-antibody complex bound to protein A-sepharose. Further evidence that the extract antigen corresponds to the stored histone pool comes from sedimentation and charge fractionation experiments where the chromatin assembly activity and antigen (measured by radioimmunoassay) were found to cofractionate. BEcause the extract histones are not bound to DNA, our results suggest that they are stored as a soluble complex in a conformation similar or identical to the octameric core of the nucleosome. Our data suggest that the histones in this complex are bound to an anionic factor or factors which presumably replaces the DNA in shielding the positive charges on the histones.