bluuebunny/crossref_metadata_2025 huggingface dataset:
Slow components of axonal transport: two cytoskeletal networks.
Authors: Black M M , Lasek R J
Published: May 2004

We have identified two slowly moving groups of axonally transported proteins in guinea pig retinal ganglion cell axons (4). The slowest group of proteins, designated slow component a (SCa), has a transport rate of 0.25 mm/d and consists of tubulin and neurofilament protein. The other slowly transported group of proteins, designated slow components b (SCb), has a transport rate of 2-3 mm/d and consists of many polypeptides, one of which is actin (4). Our analyses of the transport kinetics of the individual polypeptides of SCa and SCb indicate that (a) the polypeptides of SCa are transported coherently in the optic axons, (b) the polypeptides of SCb are also transported coherently but completely separately from the SCa polypeptides, and (c) the polypeptides of SCa differ completely from those comprising SCb. We relate these results to our general hypothesis that slow axonal transport represents the movements of structural complexes of proteins. Furthermore, it is proposed that SCa corresponds to the microtubule-neurofilament network, and that SCb represents the transport of the microfilament network together with the proteins complexed with microfilaments.
A mechanism of protein localization: the signal hypothesis and bacteria.
Authors: Emr S D , Hall M N , Silhavy T J
Published: May 2004

We are studying the molecular mechanism of cellular protein localization. The availability of genetic techniques, such as gene fusion in Escherichia coli, has made this problem particularly amenable to study in this prokaryote. We have constructed a variety of strains in which the gene coding for an outer membrane protein is fused to the gene coding for a normally cytoplasmic enzyme, beta-galactosidase. The hybrid proteins produced by such strains retain beta-galactosidase activity; this activity serves as a simple biochemical tag for studying the localization of the outer membrane protein. In addition, we have exploited phenotypes exhibited by certain fusion strains to isolate mutants that are altered in the process of protein export. Genetic and biochemical analyses of such mutants have provided evidence that the molecular mechanism of cellular protein localization is strinkingly similar in both bacteria and animal cells.
Isolation of mouse myocardial gap junctions.
Authors: Kensler R W , Goodenough D A
Published: May 2004

A new method is presented for the isolation of an enriched fraction of mouse myocardial gap junctions without the use of exogenous proteases. The junctions appear well preserved morphologically and similar to their appearance in situ. Contaminants of the preparation include fragments of the fascia adherens region of the intercalated disk. SDS polyacrylamide gel electrophoresis of the preparation reveals seven major bands with apparent mol wt of 28,000; 31,000; 33,500; 43,000; 47,000; 49,000; and 57,000. Only the bands at 38,000; 31,000; 33,500; and possibly the diffuse band at 47,000 copurify with the morphologically assayed gap junctions. Evidence is presented that the peptides at 43,000 and 57,000 are contained within the contaminating fascia adherens.
Pattern of myelin breakdown during sciatic nerve Wallerian degeneration: reversal of the order of assembly.
Authors: Patsalos P N , Bell M E , Wiggins R C
Published: May 2004

Myelin sheaths of rapidly growing rats were sequentially labeled with the 3H and 14C isotopes of leucine as precursors of protein synthesis. The two injections were separated by time intervals ranging from 2 to 12 d. Wallerian degeneration was initiated by sciatic nerve neurotomy at 2 or 10 d after the second injection of radioactivity. After 5 d of degeneration, myelin was purified and the ratio of isotopes was determined in the delipidated protein. Regardless of the order in which the two isotopes were administered, the relative recovery of radioactivity resultant from the second injection was greatly reduced in degenerating nerves compared with sham-operated controls. Radioactivity incorporated from the first injection was also reduced, but to a lesser extent. Consequently, the isotope ratio corresponding to the first/second injection was greater in degenerating nerves than in controls, and the ratio increased in proportion to the time interval separating the two injections. The magnitude of the effect of degeneration was only slightly greater when degeneration was initiated 2 d after the second injection than when initiated 10 d after the last injection. Consequently, myelin disintegration rather than diminished incorporation of radioactivity accounts for the losses of radioactivity. Furthermore, the pattern of myelin degeneration preferentially involves the last myelin to be formed.
Role of coated vesicles, microfilaments, and calmodulin in receptor-mediated endocytosis by cultured B lymphoblastoid cells.
Authors: Salisbury J L , Condeelis J S , Satir P
Published: May 2004

Cell surface receptor IgM molecules of cultured human lymlphoblastoid cells (WiL2) patch and redistribute into a cap over the Golgi region of the cell after treatment with multivalent anti-IgM antibodies. During and after the redistribution, ligand-receptor clusters are endocytosed into coated pits and coated vesicles. Morphometric analysis of the distribution of ferritin-labeled ligand at EM resolution reveals the following sequence of events in the endocytosis of cell surface IgM: (a) binding of the multivalent ligand in a diffuse cell surface distribution, (b) clustering of the ligand-receptor complexes, (c) recruitment of clathrin coats to the cytoplasmic surface of the cell membrane opposite ligand-receptor clusters, (d) assembly and (e) internalization of coated vesicles, and (f) delivery of label into a large vesicular compartment, presumably partly lysosomal. Most of the labeled ligand enters this pathway. The recruitment of clathrin coats to the membrane opposite ligand-receptor clusters is sensitive to the calmodulin-directed drug Stelazine (trifluoperazine dihydrochloride). In addition, Stelazine inhibits an alternate pathway of endocytosis that does not involve coated vesicle formation. The actin-directed drug dihydrocytochalasin B has no effect on the recruitment of clathrin to the ligand-receptor clusters and the formation of coated pits and little effect on the alternate pathway, but this drug does interfere with subsequent coated vesicle formation and it inhibits capping. Cortical microfilaments that decorate with heavy meromyosin with constant polarity are observed in association with the coated regions of the plasma membrane and with coated vesicles. SDS-polyacrylamide gel electrophoresis analysis of a coated vesicle preparation isolated from WiL2 cells demonstrates that the major polypeptides in the fraction are a 175-kdalton component that comigrates with calf brain clathrin, a 42-kdalton component that comigrates with rabbit muscle actin and a 18.5-kdalton minor component that comigrates with calmodulin as well as 110-, 70-, 55-, 36-, 30-, and 17-kdalton components. These results clarify the pathways of endocytosis in this cell and suggest functional roles for calmodulin, especially in the formation of clathrin-coated pits, and for actin microfilaments in coated vesicle formation and in capping.
Commitment of chick oviduct tubular gland cells to produce ovalbumin mRNA during hormonal withdrawal and restimulation.
Authors: Shepherd J H , Mulvihill E R , Thomas P S , Palmiter R D
Published: May 2004

Acute withdrawal of estrogen from chicks leads to a precipitous decline in egg white protein synthesis and egg white mRNAs in the oviduct. In this paper we explore the biochemical basis of this phenomenon as well as the capacity of the "withdrawn" tubular gland cells to be restimulated with steroid hormones. During withdrawal, the decline in ovalbumin mRNA was closely correlated with the decline in nuclear estrogen receptors. Within 2-3 d of estrogen removal a withdrawn state was established and then maintained, as defined by a 1,000-fold-lower level of ovalbumin mRNA and a 20-fold-lower level of nuclear estrogen receptors, relative to the estrogen-stimulated state. The number of active forms I and II RNA polymerases declined by 50% during this time. Histological examination of oviduct sections and cell suspensions, combined with measurements of DNA content, revealed that tubular gland cells persisted as a constant proportion of the cell population for 3 d after estrogen removal. Despite a 1,000-fold decrease in the content of ovalbumin mRNA, the ovalbumin gene remained preferentially sensitive to digestion by DNase I. When 3-d-withdrawn oviducts were restimulated with either estrogen or progesterone, in situ hybridization revealed that greater than or equal to 98% of the tubular gland cells contained ovalbumin mRNA. Induction by a suboptimal concentration of estrogen was correlated with a lower concentration of ovalbumin mRNA in all cells rather than fewer responsive cells.
Isolation and partial characterization of a cage of filaments that surrounds the mammalian mitotic spindle.
Authors: Zieve G W , Heidemann S R , McIntosh J R
Published: May 2004

Mitotic cells have been detergent extracted under conditions that support microtubule assembly. When HeLa cells are lysed in the presence of brain tubulin, mitotic-arrested cells nucleate large asters and true metaphase cells yield spindles that remain enclosed within a roughly spherical cage of filamentous material. Detergent-extracted mitotic Chinese hamster ovary (CHO) cells show a similar, insoluble cage but the mitotic apparatus is only occasionally stabilized. In later stages of mitosis, HeLa cages are observed in elongated and furrowed configurations. In the terminal stages of cell division, two daughter filamentous networks are connected by the intercellular bridge. When observed in the electron microscope the cages include fibers 7-11 nm in diameter. The polypeptide composition of cages isolated from mitotic HeLa cells is complex, but the major polypeptides are a group with mol wt ranging from 43,000-60,000 daltons and a high molecular weight polypeptide. CHO cells contain a subset of these proteins which includes a major 58,000-dalton and a high molecular weight polypeptide. Two different antisera directed against the vimentin-containing intermediate filaments bind to polypeptides in the electrophoretic profiles of isolated HeLa and CHO cages and stain the cages, as visualized by indirect immunofluorescence. These results suggest that the HeLa and CHO cages include intermediate filaments of the vimentin type. The polypeptide composition of HeLa cages suggests that they also contain tonofilaments. The cages apparently form as the cells enter mitosis. We propose that these filamentous cages maintain the structural continuity of the cytoplasm while the cell is in mitosis.
Structure and composition of the cytoskeleton of nucleated erythrocytes I. The presence of microtubule-associated protein 2 in the marginal band.
Authors: Sloboda R D , Dickersin K
Published: May 2004

The marginal band (MB) of nucleated erythrocytes is composed of a bundle of microtubules that encircles the cell immediately beneath the plasma membrane. When cells are lysed in buffer containing Triton X-100 the MB microtubules remain intact, and the nucleus remains suspended at the cell center by a filamentous network called the trans-MB material that connects the nucleus to the peripheral MB. When these lysed cells are prepared for indirect immunofluorescence by use of an antibody to chick brain microtubule-associated protein 2 (MAP 2), intense staining of the MB results; no staining is evident in the areas occupied by the nucleus or the trans-MB material. Controls demonstrate that the staining is specific, because no staining occurs with fluorescent goat antirabbit serum alone or when nonimmune serum is used as the first antibody. Furthermore, the fluorescence of the MB is not affected by pretreatment of the immune serum with purified tubulin, but staining is prevented by pretreatment of the immune serum with purified MAP 2. To determine which protein component of the MB was responsible for the positive immunofluorescence results, 125I-protein A staining was used after the protein components of the isolated cytoskeleton had been resolved by SDS-polyacrylamide gels. Controls showed that the antiserum could react on SDS gels with MAP 2 from purified chick brain microtubules. The results with the cytoskeletal proteins demonstrated that the antiserum reacted only with a high molecular weight protein having a molecular weight similar, but not identical, to that of chick brain MAP 2. Thus, it is concluded that a protein with antigenic characteristics similar to those of chick brain MAP 2 is a component of the MB. The results are discussed in terms of the possible function of MAP 2 in the MB.
Organelle relationships in cultured 3T3-L1 preadipocytes.
Authors: Novikoff A B , Novikoff P M , Rosen O M , Rubin C S
Published: May 2004

In differentiating 3T3-L1 cells, lipid spheres, the endoplasmic reticulum (ER), microperoxisomes, and mitochondria form "constellations" that may reflect the interplay of lipid metabolizing enzymes in these organelles. ER cisternae are also situated very close to "rosettes,"plasmalemmal specializations found in mature adipocytes in vivo. As in hepatocytes and absorptive cells of the intestine, this spatial relationship of ER and plasmalemma suggests a role for rosettes in the uptake of exogenous lipid precursors. The morphological differentiation of 3T3-L1 preadipocytes includes the loss of "stress fibers" and the appearance of microfilament like structures that encase, in a complex manner, the cytosolic lipid spheres that appear during differentiation. Other features described for the first time in 3T3-L1 preadipocytes include: (a) the presence of an extensive acid phosphatase (AcPase) positive GERL from which coated vesicles apparently arise (these coated vesicles display AcPase activity and are much smaller and far more numerous than the coated vesicles that seem to arise from the plasmalemmal coated pits); (b) the abundance of AcPase-positive autophagic vacuoles; and (c) a high level of alpha-naphthyl-acetate-esterase activity which, by light microscopy cytochemistry, appears to be localized in the cytosol.
Axonal growth during regeneration: a quantitative autoradiographic study.
Authors: Tessler A , Autilio-Gambetti A , Gambetti P
Published: May 2004

The intraaxonal distribution of labeled glycoproteins in the regenerating hypoglossal nerve of the rabbit was studied by use of quantitative electron microscope autoradiography. 9 d after nerve crush, glycoproteins were labeled by the administration of [3H]fucose to the medulla. The distribution of transported 3H-labeled glycoproteins was determined 18 h later in segments of the regenerating nerve and in the contralateral, intact nerve. At the regenerating tip, the distribution was determined both in growth cones and in non-growth cone axons, 6 and 18 h after labeling. The distribution within the non-growth cone axons of the tips was quite different at 6 and 18 h. At 6 h, the axolemma region contained < 10% of the radioactivity; at 18 h, it contained virtually all the radioactivity. In contrast, the distribution within the growth cones was similar at both time intervals, with 30% of the radioactivity over the axolemmal region. Additional segments of the regenerating nerve also showed a preferential labeling of the axolemmal region. In the intact nerve, 3H-labeled glycoproteins were uniformly distributed. These results suggest that: (a) in this system the labeled glycoproteins reaching the tip of the regenerating axons are inserted into the axolemma between 6 and 18 h after leaving the neuronal perikaryon; (b) at the times studied, there is a fairly constant ratio between glycoproteins reaching the growth cone through axoplasmic transport and glycoproteins inserted into the growth cone axolemma; (c) the axolemma elongates by continuous insertion of membrane precursors at the growth cone; the growth cone then advances, leaving behind an immature axon with a newly formed axolemma; and (d) glycoproteins are preferentially inserted into the axolemma along the entire regenerating axon.
Initiation of DNA synthesis in the prematurely condensed chromosomes of G1 cells.
Authors: Hanks S K , Rao P N
Published: May 2004

The objective of this study was to investigate whether G1 cells could enter S phase after premature chromosome condensation resulting from fusion with mitotic cells. HeLa cell synchronized in early G1, mid-G1, late G1, and G2 and human diploid fibroblasts synchronized in G0 and G1 phases were separately fused by use of UV-inactivated Sendai virus with mitotic HeLa cells. After cell fusion and premature chromosome condensation, the fused cells were incubated in culture medium containing Colcemid (0.05 micrograms/ml) and [3H]thymidine ([3H]ThdR) (0.5 microCi/ml; sp act, 6.7 Ci/mM). At 0, 2, 4, and 6 h after fusion, cell samples were taken to determine the initation of DNA synthesis in the prematurely condensed chromosomes (PCC) on the basis of their morphology and labeling index. The results of this study indicate that PCC from G0, G1, and G2 cells reach the maximum degree of compaction or condensation at 2 h after PCC induction. In addition, the G1-PCC from normal and transformed cells initiated DNA synthesis, as indicated by their "pulverized" appearance and incorporation of [3H]ThdR. Further, the initiation of DNA synthesis in G1-PCC occurred significantly earlier than in the mononucleate G1 cells. Neither pulverization nor incorporation of label was observed in the PCC of G0 and G2 cells. These findings suggest that chromosome decondensation, although not controlling the timing of a cell's entry into S phase, is an important step for the initiation of DNA synthesis. These data also suggest that the entry of a S phase may be regulated by cell cycle phase-specific changes in the permeability of the nuclear envelope to the inducers of DNA synthesis present in the cytoplasm.
Establishment and transformation diminish the ability of fibroblasts to contract a native collagen gel.
Authors: Steinberg B M , Smith K , Colozzo M , Pollack R
Published: May 2004

Cultures of established and transformed fibroblasts were less able to contract a hydrated collagen gel than normal precrisis cells. Postcrisis fibroblasts from different rodent strains and species underwent a further reduction in contraction ability and either spontaneous or simian virus 40 (SV40) transformation. Human precrisis fibroblasts contracted much more efficiently than two SV40-transformed human lines. Fibroblasts from a patient with Glanzmann's thrombasthenia were intermediate between all other human fibroblasts assayed and the SV40-transformed human lines. The absolute efficiency of contraction was dependent on temperature and serum concentration, but no conditions were found that resulted in equal efficiencies for the three types of cells. Precrisis cells were extremely sensitive to the passage procedures when assayed for collagen contraction.
Fibronectin in the developing sea urchin embryo.
Authors: Spiegel E , Burger M , Spiegel M
Published: May 2004

The presence of fibronectin in developing sea urchin embryos was studied uing immunofluorescence staining. The fluorescence pattern indicates that fibronectin is found on the cell surfaces and between cells in the blastula and gastrula stages, indicating that it plays a role in cell adhesion. Its presence on invaginating cells also suggests its involvement in morphogenesis during early development.
Cytoplasmic microtubules and fungal morphogenesis: ultrastructural effects of methyl benzimidazole-2-ylcarbamate determined by freeze-substitution of hyphal tip cells.
Authors: Howard R J , Aist J R
Published: May 2004

The effects of methyl benzimidazole-2-ylcarbamate (MBC), one of only a few agents that are active against microtubules of fungi, were analyzed at the ultrastructural level in freeze-substituted hyphal tip cells of Fusarium acuminatum. Nontreated and control cells had numerous microtubules throughout. After just 10 min of exposure to MBC, almost no cytoplasmic microtubules were present, except near spindle pole bodies. After 45 min of exposure to MBC, no microtubules were present in hyphal tip cells, but they were present in the relatively quiescent subapical cells. These observations suggested that there are different rates of turnover for cytoplasmic microtubules in apical and subapical cells and for microtubules near spindle pole bodies and that MBC acts by inhibiting microtubules assembly. A statistical analysis of the distribution of intracytoplasmic vesicles in thick sections of cells treated with MBC, D2O or MBC + D2O was obtained by use of a high-voltage electron microscope. More than 50% of the vesicles in the apical 30 micrometers of control cells were found to lie within 2 micrometers of the tip cell apex. MBC treatment caused this vesicle distribution to become uniform, resulting in a substantial increase in the number of vesicles in subapical regions. The reduction in the number of cytoplasmic microtubules, induced by MBC, apparently inhibited intracellular transport of these vesicles and rendered random the longitudinal orientation of mitochondria. In most cases, D2O appeared capable of preventing these MBC-effects through stabilization of microtubules. These observations support the "vesicle hypothesis" of tip growth and establish a transport role for cytoplasmic microtubules in fungal morphogenesis.
Analytical characterization of beetroot vacuole membrane.
Authors: Marty F , Branton D
Published: May 2004

Vacuoles from beetroot (Beta vulgaris L. var. esculenta Gürke) isolated by a mechanical procedure were osmotically lysed to separate the membrane and sap components for analysis. Approximately 62% of the vacuole proteins, 70% of the nondialyzable carbohydrates and almost all of the phospholipids and sterols were recovered in the membrane fraction. The vacuole membrane had a phospholipid protein ratio of 0.68 and a sterol:phospholipid ratio of 0.21. 17 complex polar lipids including phosphatides and glycolipids have been tentatively identified. Phosphatidylcholine (54%) and phosphatidylethanolamine (24%) were the most prominent phosphoglycerides besides phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid (1, 4, 5, and 12%, respectively). A putative sulfoglycoside and two major ceramide glycoside-like lipids, resembling those of animal lysosomes, were identified by thin-layer chromatography. High-resolution SDS-acrylamide gel electrophoresis of the polypeptides from the vacuole revealed 15 major bands with apparent molecular weights ranging from 91,000 to 12,000. Selective elution experiments delineated those polypeptides that were peripheral membrane proteins or sap proteins adsorbed to the membrane, and those that exhibited hydrophobic interactions with the lipid core. Lectin labeling results indicated that most of the polypeptides from the membrane and from the sap were glycoproteins probably of the high-mannose type characteristic of lysosomal enzymes that have undergone several stages of posttranslational modification.
Interactions of dynein arms with b subfibers of Tetrahymena cilia: quantitation of the effects of magnesium and adenosine triphosphate.
Authors: Mitchell D R , Warner F D
Published: May 2004

Tetrahymena 30S dynein was extracted with 0.5 M KCl and tested for retention of several functional properties associated wtih its in situ force-generating capacity. The dynein fraction will rebind to extracted outer doublets in the presence of Mg2+ to restore dynein arms. The arms attach at one end to the A subfiber and form bridges at the other end to the B subfiber of an adjacent doublet. Recombined arms retain an ATPase activity that remains coupled to potential generation of interdoublet sliding forces. To examine important aspects of the dynein-tubulin interaction that we presume are directly related to the dynein force-generating cross-bridge cycle, a simple and quantitative spectrophotometric assay was devised for monitoring the associations between isolated 30S dynein and the B subfiber. Utilizing this assay, the binding of dynein to B subfibers was found to be dependent upon divalent cations, saturating at 3 mM Mg2+. Micromolar concentrations of MgATP2- cause the release of dynein from the B subfiber; however, not all of the dynein bound under these conditions is released by ATP. ATP-insensitive dynein binding results from dynein interactions with non-B-tubule sites on outer-doublet and central-pair microtubules and from ATP-insensitive binding to sites on the B subfiber. Vanadate over a wide concentration range (10(-6)-10(-3) M) has no effect on the Mg2+-induced binding of dynein or its release by MgATP2-, and was used to inhibit secondary doublet disintegration in the suspensions. In the presence of 10 microM vanadate, dynein is maximally dissociated by MgATP2- concentrations greater than or equal to 1 microM with half-maximal release at 0.2 microM. These binding properties of isolated dynein arms closely resemble the cross-bridging behavior of in situ dynein arms reported previously, suggesting that quantitative studies such as those presented here may yield reliable information concerning the mechanism of force generation in dynein-microtubule motile systems. The results also suggest that vanadate may interact with an enzyme-product complex that has a low affinity for tubulin.
An antiserum specific for cholinergic synaptic vesicles from electric organ.
Authors: Carlson S S , Kelly R B
Published: May 2004

Rabbit antisera to highly purified synaptic vesicles from the electric organ of Narcine brasiliensis, an electric ray, reveal a unique population of synaptic vesicle antigens in addition to a population shared with other electric organ membranes. Synaptic vesicle antigens were detected by binding successively rabbit antivesicle serum and radioactive goat anti-rabbit serum. To remove antibodies directed against antigens common to synaptic vesicles and other electric organ fractions, the antivesicle serum was extensively preadsorbed against an electric organ membrane fraction that was essentially free of synaptic vesicles. The adsorbed serum retained 40% of its ability to bind to synaptic vesicles, suggesting that about half of the antigenic determinants are unique. Vesicle antigens were quantified with a radioimmunoassay (RIA) that utilized precipitation of antibody-antigen complexes with Staphylococcus aureus cells. By this assay, the vesicles, detected by their acetylcholine (ACh) content and the antigens detected by the RIA, have the same buoyant density after isopycnic centrifugation of crude membrane fractions on sucrose and glycerol density gradients. The ratio of ACh to antigenicity was constant across the vesicle peaks and was close to that observed for vesicles purified to homogeneity. Even though the vesicles make up only approximately 0.5% of the material in the original homogenate, the ratio of acetylcholine to vesicle antigenicity could still be measured and also was indistinguishable from that of pure vesicles. We conclude that synaptic vesicles contain unique antigenic determinants not present to any measurable extent in other fractions of the electric organ. Consequently, it is possible to raise a synaptic vesicle-specific antiserum that allows vesicles to be detected and quantified. These findings are consistent with earlier immunohistochemical observations of specific antibody binding to motor nerve terminals.
Immunofluorescent localization of cyclic nucleotide-dependent protein kinases on the mitotic apparatus of cultured cells.
Authors: Browne C L , Lockwood A H , Su J L , Beavo J A , Steiner A L
Published: May 2004

Cyclic nucleotides and cyclic nucleotide-dependent protein kinases have been implicated in the regulation of cell motility and division, processes that depend on the cell cytoskeleton. To determine whether cyclic nucleotides or their kinases are physically associated with the cytoskeleton during cell division, fluorescently labeled antibodies directed against cyclic AMP, cyclic GMP, and the cyclic nucleotide-dpendent protein kinases were used to localize these molecules in mitotic PtK1 cells. Both the cyclic GMP-dependent protein kinase and the type II regulatory subunit of the cyclic AMP-dependent protein kinase were localized on the mitotic spindle. Throughout mitosis, their distribution closely resembled that of tubulin. Antibodies to cyclic AMP, cyclic GMP, and the type I regulatory and catalytic subunits of the cyclic AMP-dependent protein kinase did not label the mitotic apparatus. The association between specific components of the cyclic neucleotide system and the mitotic spindle suggests that cyclic nucleotide-dependent phosphorylation of spindle proteins, such as those of microtubules, may play a fundamental role in the regulation of spindle assembly and chromosome motion.
Effects of fasciculation on the outgrowth of neurites from spinal ganglia in culture.
Authors: Rutishauser U , Edelman G M
Published: May 2004

This report describes the influence of neurite fasciculation on two aspects of nerve growth from chick spinal ganglia in vitro: the inhibition of outgrowth by high concentrations of nerve growth factor (NGF) and the preferential growth of neurites toward a capillary tube containing NGF. These studies involved a comparison of cultures of single cells, cell aggregates, and intact ganglia and the use of antibodies against the nerve cell adhesion molecule (CAM) to perturb fasciculation under a variety of conditions. The inhibition of outgrowth, which was observed with ganglia and aggregates but not with single cells, was correlated with a thickening of neurite fascicles. In accord with this observation, anti-CAM, which diminishes fasciculation by inhibiting side-to-side interactions between individual neurites, also partially reversed the inhibition of neurite outgrowth at high NGF concentrations. On the basis of these and other studies, we consider the possibility that neurite bundling causes an increase in the elastic tension of a fascicle without a compensatory increase in its adhesion to substratum. It is proposed that this imbalance could inhibit neurites from growing out from a ganglion and even result in retraction of preexisting outgrowth. In the analysis of NGF-directed growth, it was found that a capillary source of NGF produced a steep but transient NGF gradient that subsided before most neurites had emerged from the ganglion. Nevertheless, the presence of a single NGF capillary caused a dramatic and persistent asymmetry in the outgrowth of neurites from ganglia or cell aggregates. In contrast, processes of individual cells did not appear to orient themselves toward the capillary. The most revealing finding was that anti-CAM antibodies caused a decrease in the asymmetry of neurite outgrowth. These results suggest that side-to-side interactions among neurites can influence the guidance of nerve bundles by sustaining and amplifying an initial directional signal.
Evidence for multiple somatic pools of individual axonally transported proteins.
Authors: Berry R W
Published: May 2004

The idea that individual axonally transported proteins can exist in several kinetically distinct pools within the cell body was studied using the presumptive neurosecretory low molecular weight (LMW) proteins of Aplysia neurons L11 and R15. Pulse-chase experiments revealed that the loss of labeled LMW proteins from the soma by axonal transport does not follow single exponential kinetics as it should if they are being removed from single pools. Rather, decay of label occurs in at least two phases having half-lives of approximately 1 and 40 h. The LMW proteins are homogeneous by sequential SDS gel electrophoresis and isoelectric focusing, indicating that individual protein species exhibit multiphasic decay kinetics. Two types of evidence imply that the bulk of cellular LMW protein turns over at the slower rate: the LMW pool does not reach constant specific activity at the rapid rate during continuous exposure to labeled precursor, and long-term blockade of axonal transport does not produce an appreciable accumulation of these species in the cell body. These results suggest that some of the newly synthesized LMW protein enters a small somatic pool from which it is rapidly subjected to axonal transport, while the remainder enters a larger pool where it can mix with previously synthesized protein before transport. A cellular mechanism that would yield this behavior is suggested.
A selective effect of Ni2+ on wave initiation in bull sperm flagella.
Authors: Lindemann C B , Fentie I , Rikmenspoel R
Published: May 2004

Bull sperm that are extracted with 0.1% Triton X-100 and restored to motility with Mg2+-ATP lose coordination and stop swimming in the presence of 0.5 mM NiSO4. Although spontaneous coordination of flagellar waves is lost after exposure to Ni2+, other functions of the flagellum remain intact. The capacity for wave propagation along the flagellum is maintained together with the capacity for microtubular sliding. Wave motility can be restored to Ni2+-inhibited sperm by inducing a permanent bend onto the flagellum by micromanipulation. In the absence of such intervention, the loss of wave coordination is complete and irreversible. Ni2+-inhibited demembranated cells that are kept active by maintaining a bend in the flagellum exhibit a normal beat frequency. Both intact and demembranated sperm can retain spontaneous wave production at considerably slower rates of motion than Ni2+-inhibited cells. Short segments from the distal tip of the flagellum contain only the 9 + 2 microtubular axoneme. These short segments are able to propagate imposed bends even in the presence of Ni2+. In addition to wave propagation Ni2+-treated sperm can be shown to exhibit a normal sliding tubule phenomenon by direct assay. Although Ni2+-treated cells have a functional sliding tubule mechanism, and consequently the axoneme can propagate bends, it appears that these retained functions are not sufficient to cause spontaneous bend initiation. Our findings show that bend initiation is inhibited by Ni2+, and therefore is an independent process separate from the sliding tubule mechanism responsible for wave propagation.
Polarity of midbody and phragmoplast microtubules.
Authors: Euteneuer U , McIntosh J R
Published: May 2004

A newly discovered method (Heidemann and McIntosh, 1980, Nature [Lond.] 286:517) for displaying the molecular polarity of microtubules (MTs) has been slightly modified and applied to the midbodies of cultured mammalian cells and the phragmoplasts of Haemanthus endosperm. The method involves the decoration of preexisting MTs in lysed cells with curved ribbons of tubulin protofilaments; the direction of curvature of these C-shaped appendages as seen in cross section reflects the intrinsic polarity of the MTs. In travsverse sections of midbodies from HeLa and PtK cells, we find that essentially all the MTs in a given region of the structures have the same direction of hook curvature, and hence the same polarity. The midbody MTs that lie on one side of the spindle equator show the opposite polarity from those on the other side, indicating that the midbody is constructed from two families of antiparallel MTs. Midbody MTs are arranged with their fast-growing ends overlapping at the spindle equator, consistent with the hypothesis that the midbody is formed by the interdigitation of aster MTs. The polarities of the MTs from the phragmoplast of endosperm cells are the same as those found in the mammalian midbody. Our results eliminate one model for mitosis, but are consistent with others. The systematic and reproducible polarities observed favor the concept that MT polarity is an important factor in the formation and/or the function of these two mitotic structures.
Signal peptides and signal peptidase in Drosophila melanogaster.
Authors: Brennan M D , Warren T G , Mahowald A P
Published: May 2004

Translation of poly(A)-containing RNA from the female fat body of Drosophila melanogaster in a rabbit reticulocyte cell-free system results in the synthesis of previtellogenin polypeptides (PVs) having higher apparent molecular weights (46,000 and 45,000) than the forms seen after an in vivo pulse labeling. However, when this RNA is translated in the presence of EDTA-stripped microsomal membranes from the dog pancreas, vitellogenin precursors are produced that, upon SDS-polyacrylamide gel electrophoresis, comigrate with the in vivo forms (apparent molecular weights, 45,000 and 44,000). These processed forms are sequestered within the microsomal lumen, as evidenced by their insensitivity to trypsin digestion. Neither processing nor sequestration occur posttranslationally. In addition, a microsomal membrane fraction derived from Drosophila embryos is able to cotranslationally process the PVs as well as a murine pre-light chain IgG. These observations support a signal-mediated mode of secretion in Drosophila, and suggest that signal sequence recognition and signal peptidase activities are conserved even between mammalian and insect systems.
Characterization of apolipoprotein B from human serum low density lipoprotein in n-dodecyl octaethyleneglycol monoether: an electron microscope study.
Authors: Zampighi G , Reynolds J A , Watt R M
Published: May 2004

We have studied the structure of the totally delipidated polypeptide (apolipoprotein B [apo B]) present in low-density serum lipoprotein in detergent (n-dodecyl octaethyleneglycol monoether) solution by electron microscopy. The protein-detergent complex appears as a rod-shaped particle, 75-80 nm long and 4.5-5.5 nm wide. The volume of this particle is consistent with the previously published composition reported by Watt and Reynolds (1980, Biochemistry 19:1593-1598) of two copies of apo B and five to six equivalent micelles of detergent. The asymmetric particle possesses a high degree of flexibility and a strong tendency to self-associate in an orderly fashion. The extent of this association is pH dependent.
Specializations in filopodial membranes at points of attachment to the substrate.
Authors: Robinson J M , Karnovsky M J
Published: May 2004

A mouse cell line (LM), which grows predominantly as spindle-shaped cells with numerous filopodia, was employed in this study. These filopodial projections appear to be important as sites of attachment to the substratum in LM cells. Morphologically the filopodia are slender projections from the cell body which usually attach to the substrate at their distal ends (filopodial footpads). Freeze-fracture of monolayer cultures in situ preserves the spatial relationship of filopodial processes to that of the cell body. Examination of these freeze-fracture preparations reveals a striking difference in the density of intramembrane particles (IMP) in the filopodial-footpad plasmalemma compared with the plasmalemma of the cell body (number of IMP in footpad > cell body). Additionally, there is a marked difference in the number of filipin-sterol complexes on the cell body, compared with the filopodial footpad, implying a difference in the cholesterol content in these regions (filipin-sterol complexes in footpad < cell body). These data suggest a structural and functional specialization of the filopodial-footpad plasma membrane which may be related to cell adhesion.
Pathways involved in fluid phase and adsorptive endocytosis in neuroblastoma
Authors: Gonatas J , Steiber A , Olsnes S , Gonatas NK
Published: May 2004

The endocytosis of ricin, horseradish peroxidase (HRP), and a conjugate of ricin-HRP by monolayer cultures of murine neuroblastoma was studied using morphological and biochemical techniques.

The binding of (125)I-ricin and (125)I-ricin-HRP to cells at 4 degrees C, as a function of ligand concentration, was a saturable process. The apparent affinity constants, determined at equilibrium, were 2.8 X 10(6) M(-1) for ricin and 1 x 10(6) M(-1) for ricin-HRP. The number of binding sites per cell was 8 x 10(7) and 3 x 10(7) for the lectin and the conjugate, respectively. The binding of (125)I-ricin to monolayers as not proportional to cell density. We found reduced binding at higher cell concentrations, suggesting a decrease in the accessibility of the ligand for the receptor site or fewer sites with increasing cell population.

Neuroblastoma cells have an acid-phosphatase-positive network of cisternae and vesicles near the Golgi apparatus (GERL). Ricin-HRP undergoes endocytosis in vesicles and cisternae corresponding to GERL, and in residual bodies (dense bodies). The cellular uptake of ricin-HRP was 100-200 times greater than free HRP and there was no stimulation of fluid phase endocytosis by ricin. When monolayers were exposed to concentrations of native HRP 100-fold that of the conjugate, cellular uptake of peroxidase was comparable, but HRP was localized only in residual bodies and never in elements of GERL. These results support the conclusion that GERL is involved in the adsorptive endocytosis of ricin-HRP, while residual bodies are involved in the bulk uptake of HRP.

In addition, the binding, uptake, and possible recycling of (125)I- subunit B (the binding subunit) of ricin and of (125)I-ricin was examined by quantitative electron microscope autoradiography. Both ricin and its binding subunit displayed similar autoradiographic grain distributions at 4 degrees C, and there was no evidence of their breakdown or recycling to the plasma membrane during endocytosis for 2 h.
Immunocytochemical localization of procollagen and fibronectin in human fibroblasts: effects of the monovalent ionophore, monensin.
Authors: Ledger P W , Uchida N , Tanzer M L
Published: May 2004

The monovalent ionophore monensin inhibits the secretion of both procollagen and fibronectin from human fibroblasts in culture. The distribution of these proteins in control and inhibited (5 x 10(-7) M monensin) cells has been studied by immunofluorescence microscopy. In control cells, both antigens are present throughout the cytoplasm and in specific deposits in a region adjacent to the nucleus, which we identify as a Golgi zone by electron microscopy. Treatment of cells with monensin causes intracellular accumulation of procollagen and fibronectin, initially in the juxta-nuclear region and also subsequently in peripheral regions. Electron microscope studies reveal that in such cells the juxta-nuclear Golgi zone becomes filled with a new population of smooth-membraned vacuoles and that normal Golgi complexes are not found. Immunocytochemically detected procollagen and fibronectin are localized in the region of these vacuoles, whereas more peripheral deposits correspond to the dilated cisternae of rough endoplasmic reticulum, which are also caused by monensin. Procollagen and fibronectin are often codistributed in these peripheral deposits. Accumulation of exportable proteins in Golgi-related vacuoles is consistent with previous analyses of the monensin effect. The subsequent development of dilated rough endoplasmic reticulum also containing accumulated proteins may indicate that there is an additional blockade at the exit from the endoplasmic reticulum, or that the synthesized proteins exceed the capacity of the Golgi compartment and that their accumulation extends into the endoplasmic reticulum.
Separation of renal medullary cells: isolation of cells from the thick ascending limb of Henle's loop.
Authors: Eveloff J , Haase W , Kinne R
Published: May 2004

A homogeneous population of single cells from the thick ascending limb of Henle's loop (TALH) has been isolated from the rabbit kidney medulla. A total medullary cell suspension was prepared by a series of collagenase, hyaluronidase, and trypsin digestions and separated on a Ficoll gradient (2.6-30.7% wt/wt). Morphologically, the cells isolated from the TALH were homogeneous and showed polarity within their plasma membrane structure, with a few blunt microvilli on their apical surface and deep infoldings of the basal-lateral membrane. Biochemically, the TALH cells were highly enriched in calcitonin-sensitive adenylate cyclase and Na, K-ATPase. Alkaline phosphatase and arginine vasopressin-sensitive adenylate cyclase, highly concentrated in proximal tubule and collecting duct, were present only in low concentrations in the TALH cells. Additionally, furosemide, a diuretic inhibiting sodium chloride transport in the TALH in vivo, inhibited oxygen consumption of the TALH cells in a dose-dependent manner. The TALH cells were viable, as judged by morphological appearance, trypan blue exclusion, the response of oxygen consumption to 2,4-dinitrophenol, succinate and ouabain, and the cellular Na, K and ATP levels.
Actin, microvilli, and the fertilization cone of sea urchin eggs.
Authors: Tilney L G , Jaffe L A
Published: May 2004

Sea urchin eggs and oocytes at the germinal vesicle stage were fixed at various times after insemination, and thin sections were examined. Actin filaments can first be found in the cortical cytoplasm 1 min after insemination, and by 2 min enormous numbers of filaments are present. At these early stages, the filaments are only occasionally organized into bundles, but one end of many filaments contacts the plasma membrane. By 3 min, and even more dramatically by 5 min after insemination, the filaments become progressively more often found in bundles that lie parallel to the long axis of the microvilli and the fertilization cones. By 7 min, the bundles of filaments in the cone are maximally pronounced, with virtually all the filaments lying parallel to one another. Decoration of the filaments with subfragment 1 of myosin shows that, in both the microvilli and the cones, the filaments are unidirectionally polarized with the arrowheads pointing towards the cell center. The efflux of H+ from the eggs was measured as a function of time after insemination. The rapid phase of H+ efflux occurs at the same time as actin polymerization. From these results it appears that the formation of bundles of actin filaments in microvilli and in cones is a two-step process, involving actin polymerization to form filaments, randomly oriented but in most cases having one end in contact with the plasma membrane, followed by the zippering together of the filaments by macromolecular bridges.
Immunofluorescence localization of HeLa cell microtubule-associated proteins on microtubules in vitro and in vivo.
Authors: Bulinski J C , Borisy G G
Published: May 2004

Rabbit antisera were prepared against the two major groups of microtubule-associated proteins (MAPs) from HeLa cells, proteins of approximately 210,000 molecular weight (210k MAPs), and 125,000 mol wt (125k MAPs). These antisera were characterized by a sensitive antigen detection technique that employs immunofluorescence to localize cross-reactive material in polyacrylamide gels. Antisera prepared against the 210k MAPs showed no cross-reactivity with extract proteins of other molecular weights or with bran MAPs, but did react with proteins of 210,000 mol wt and with a minor HeLa MAP of approximately 255,000 mol wt. Antibodies prepared against the 125k HeLa MAPs, likewise, reacted specifically with proteins of 125,000 mol wt, showing no cross-reactivity with other HeLa extract proteins or porcine brain MAPs. Immunofluorescence with the 210k and 125k MAP antisera was used to demonstrate the association of each of the MAPs with fixed HeLa microtubules in vitro. In addition, immunofluorescence with these antisera revealed a physical association of 210k and 125k MAPs with a Colcemid-sensitive fiber network in fixed interphase and mitotic HeLa cells. Thus, using specific, well-characterized antisera to the two major groups of HeLa MAPs, we have shown that these proteins are components of microtubules in HeLa cells.