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S R Heidemann

Publications and source records attributed to S R Heidemann.

53 records · Page 3Linked to original sources
PubMed1985

Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and microtubule stability of PC12 cells.

The outgrowth of neurites from rat PC12 cells stimulated by combined treatment of nerve growth factor (NGF) with cAMP is significantly more rapid and extensive than the outgrowth induced by either factor alone. We have compared the responses of PC12 cells under three different growth conditions, NGF alone, cAMP alone, and combined treatment, with respect to surface morphology, rapidity of neurite outgrowth, and stability of neurite microtubules, to understand the synergistic action of NGF and cAMP on PC12. Surface events at early times in these growth conditions varied, suggesting divergent pathways of action of NGF and cAMP. This suggestion is strongly supported by the finding that cells exposed to saturating levels of dibutyryl cAMP without substantial neurite outgrowth initiated neurites within 5 min of NGF. This response has been adopted as a convenient assay for NGF. Neurites that regenerated in the three growth conditions showed marked differences in stability to treatments that depolymerize microtubules. The results indicate that microtubules in cells treated with both NGF and cAMP are significantly more stable than in either growth factor alone. We suggest that a shift of the assembly equilibrium favoring tubulin assembly is a necessary prerequisite for the initiation of neurites by PC12.

Animals↗
PubMed1985

Tension and compression in the cytoskeleton of PC 12 neurites.

We report in this article that the retraction of PC 12 neurites, unlike that of other cultured neurons, is due to tension within the neurite. Retraction is rapid and independent of metabolic energy. Transection of one arm of a branched neurite immediately causes the remaining arm to take up a new equilibrium position between attachment points. Similarly, detachment of one growth cone of a cell causes the cell body to move to a new equilibrium position between the remaining neurites. These observations provide direct evidence for the suspension of the cell soma among a network of tensioned neurites. We used retraction as an assay for neurite tension to examine the role of actin filaments and microtubules in neurite support and elongation. Our data suggest that microtubules (MTs) within PC 12 neurites are under compression, supporting tension within the actin network. Treatment of cells with drugs that disrupt actin networks, cytochalasin D or erythro-9-[3-(2-hydroxynonyl)]adenosine eliminates retraction regardless of the absence of MTs, lack of adhesion to the substratum, or integrity of the neurite. Conversely, stimulation of actin polymerization by injection of phalloidin causes retraction of neurites. Treatments that depolymerize MTs, nocodazole or cold, cause retraction of neurites, which suggests that microtubules support this tension, i.e., are under compression. Stabilization of MTs with taxol stabilizes neurites to retraction and under appropriate circumstances can drive neurite extension. Taxol-stimulated neurite extension is augmented by combined treatment with anti-actin drugs. This is consistent with the actin network's normally exerting a force opposite that of MT assembly. Cytochalasin and erythro-9-[3-(2-hydroxynonyl)] adenosine were found to increase slightly the dose of nocodazole required for MT depolymerization. This is consistent with the postulated balance of forces and also suggests that alteration of the compression borne by the microtubules could serve as a local regulator for MT polymerization during neurite outgrowth.

Actins↗
PubMed1985

Microtubules in immature oocytes of Xenopus laevis.

Previous work indicated that immature oocytes of Xenopus were incapable of assembling microtubules but that competence was achieved during maturation. We report here that small numbers of microtubules do exist in immature oocytes. Consistent with this finding, ultrastructural observations indicate that brain microtubules injected into immature oocytes persist in large numbers for at least 30 min. We report that the tubulin dimers of mature and immature oocytes are equally capable of assembling with brain tubulin in vitro. We confirmed previous results that injection of taxol into immature oocytes has no effect when assayed by light microscopy. However, ultrastructural observations suggest that some microtubule assembly is stimulated by taxol. We tested for the ability of immature oocytes to elongate microtubules from 'seeds' by injecting deciliated pellicles of Tetrahymena. No elongation was observed either by light or electron microscopic observation. We conclude that the immature oocyte is capable of very limited microtubule assembly and that a marked increase in assembly competence occurs during maturation. Our data suggest that the change in assembly competence during maturation is due to the release, activation or synthesis of a stimulatory co-factor.

Alkaloids↗
PubMed1984

Cardiac atria of BIO 14.6 hamsters are deficient in natriuretic factor.

The hearts of 220-day-old hamsters of the BIO 14.6 strain are deficient in atrial natriuretic factor; saline extracts of atria produce one-third the natriuretic and diuretic effects of extracts of atria from age-matched normal hamsters. BIO 14.6 hamsters are known to develop congestive heart failure with edema when they are about 200 days old, and the venous congestion and edema are preventable by parabiosis with normal hamsters. The humoral mediator, the deficiency of which causes venous congestion and edema in BIO 14.6 hamsters, may be atrial natriuretic factor.

Animals↗
PubMed1984

The thiol-disulfide balance during maturation of Xenopus laevis oocytes.

We measured several of the components involved in the thiol-disulfide balance of immature and mature oocytes of Xenopus laevis: total soluble native sulfhydryl, reduced and oxidized glutathione, native sulfhydryl content of soluble protein, and disulfide content of soluble protein. None of these values changed during maturation. Both the immature and mature oocyte were characterized by a relatively high disulfide to sulfhydryl ratio. We also measured native sulfhydryl content of germinal vesicle proteins and enucleated oocyte proteins in immature oocytes. Contrary to early cytochemical findings, our measurements suggest that the germinal vesicle has a lower concentration of protein sulfhydryl than does the cytoplasm.

Animals↗
PubMed1984

Spatial organization of axonal microtubules.

Several workers have found that axonal microtubules have a uniform polarity orientation. It is the "+" end of the polymer that is distal to the cell body. The experiments reported here investigate whether this high degree of organization can be accounted for on the basis of structures or mechanisms within the axon. Substantial depolymerization of axonal microtubules was observed in isolated, postganglionic sympathetic nerve fibers of the cat subjected to cold treatment; generally less than 10% of the original number of microtubules/micron 2 remained in cross section. The number of cold stable MTs that remained was not correlated with axonal area and they were also found within Schwann cells. Microtubules were allowed to repolymerize and the polarity orientation of the reassembled microtubules was determined. In fibers from four cats, a majority of reassembled microtubules returned with the original polarity orientation. However, in no case was the polarity orientation as uniform as the original organization. The degree to which the original orientation returned in a fiber was correlated with the number of cold-stable microtubules in the fiber. We suggest that stable microtubule fragments serve as nucleating elements for microtubule assembly and play a role in the spatial organization of neuronal microtubules. The extremely rapid reassembly of microtubules that we observed, returning to near control levels within the first 5 min, supports microtubule elongation from a nucleus. However, in three of four fibers examined this initial assembly was followed by an equally rapid, but transient decline in microtubule number to a value that was significantly different than the initial peak. This observation is difficult to interpret; however, a similar transient peak has been reported upon repolymerization of spindle microtubules after pressure induced depolymerization.

Animals↗
PubMed1984

Splenic and renal sympathetic responses to stimulation of splenic receptors in cats.

The effect of selective stimulation of splenic receptors on reflex responses of splenic and renal efferent nerves was studied in anesthetized, vagotomized, sinoaortic denervated cats. The following substances were injected into the artery or vein of vascularly isolated spleens: warm physiological saline (congestion), capsaicin (CAPS), bradykinin (BK), and norepinephrine (NE). Splenic congestion increased efferent activity of splenic and renal nerves, splenic venous pressure, systemic arterial pressure, and heart rate. CAPS and BK elicited responses similar to those produced by congestion but caused greater excitation of splenic than renal nerves. Reflex responses were eliminated by section of splenic nerves. Injection of NE increased splenic venous pressure but did not elicit reflex responses. Finally, in contrast to a previous report light- and electron-microscopic examination of splenic nerves revealed myelinated, as well as unmyelinated, fibers. These results have demonstrated that activation of splenic receptors elicits reflex cardiovascular responses and excitation of splenic and renal efferent nerves, contraction of the spleen does not produce reflex responses, the adequate stimulus for reflex responses is stretch of vessels and possibly capsule, and splenic receptors play a role in the reflex control of circulation.

Animals↗
PubMed1981

Polarity orientation of axonal microtubules.

The polarity orientation of cellular microtubules is widely regarded to be important in understanding the control of microtubule assembly and microtubule-based motility in vivo. We have used a modification of the method of Heidemann and McIntosh (Nature (Lond.). 286:517-519) to determine the polarity orientation of axonal microtubules in postganglionic sympathetic fibers of the cat. In fibers from three cats we were able to visualize the polarity of 68% of the axonal microtubules; of these, 96% showed the same polarity orientation. Our interpretation is that the rapidly growing end of all axonal microtubules is distal to the cell body. We support Kirschner's hypothesis on microtubule organizing centers. (J. Cell Biol. 86:330-334), although this interpretation raises questions about the continuity of axonal microtubules. Our results are inconsistent with a number of models for axonal transport based on force production on the surface of microtubules in which the direction of force is determined by the polarity of microtubules.

Animals↗
PubMed1980

Visualization of the structural polarity of microtubules.

Microtubules (MTs) are intrinsically polar fibres. Many models for the mechanism of MT-mediated transport and mitosis postulate an important role for MT polarity because a symmetric fibre could not generate force in one direction along its surface. The lack of a convenient method to determine the polar orientation of cellular MTs has hindered experimental testing of these ideas. We report here a convenient technique for visualizing MT polarity based on conditions for tubulin assembly in vitro whereby MT elongation occurs with the associated formation of hooked, protofilament appendages on the MT wall (see Fig. 1). There is polarity information in such junctions between MT walls; our method reveals the polarity of the cellular Mt by the handedness of the hooked appendage as seen in cross-section (Fig. 1).

Animals↗
PubMed1980

Evidence for microtubule subunit addition to the distal end of mitotic structures in vitro.

HeLa cells blocked in metaphase with 0.04 micrograms/ml of the microtubule poison nocodazole were shown to contain large numbers of microtubules with typical mitotic organization but no cenriole. Lysis of nocodazole-poisoned cells in a microtubule reassembly buffer containing 0.5 M PIPES, 2.5% dimethyl sulfoxide, 1 mM EDTA, 1 mM MgCl2, 1 mM GTP, 1% Triton X-165, 0.5% sodium deoxycholate, 0.2% SDS, pH 6.9, preserved metaphase aster structures 5 micrograms in diameter surrounded only by a thin, fibrous cell remnant. Inclusion of 2 mg/ml porcine brain microtubule protein in the lysis buffer produced asters up to 20 micrometers in diameter with a birefringent retardation of 5-6 nm. In these large asters the central microtubules had normal morphology, but peripheral microtubules were clearly abnormal. Our interpretation is that in high PIPES lysis buffer, exogenous brain tubulin adds to the distal ends of preexisting aster microtubules to form abnormal microtubules. This observation supports the assumptions made by Borisy and by Summers and Kirschner in their interpretation of growth experiments to determine the microtubule polarity in mitotic structures.

Benzimidazoles↗
PubMed1980

Isolation and partial characterization of a cage of filaments that surrounds the mammalian mitotic spindle.

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.

Animals↗
PubMed1978

Induced formation of asters and cleavage furrows in oocytes of Xenopus laevis during in vitro maturation.

We have previously reported that injection of purified basal bodies or sperm into unfertilized eggs of Xenopus laevis induced the formation of asters and irregular cleavage furrows. Fully grown oocytes were found to be unable to form asters or cleavage furrows. In this paper we show that the oocyte acquires the ability to form asters upon basal body injection at the time of germinal vesicle breakdown during in vitro maturation. Our evidence indicates that aster formation requires progesterone-stimulated changes in the oocyte and mixing of cytoplasm and germinal vesicle plasm. The ability of the oocyte to form cleavage furrows arises six to eight hours after germinal vesicle breakdown. We infer that some maturational change in the cell cortex occurs to enable the egg surface to furrow. Experiments on the relationship of aster formation to furrow initiation indicates that asters stimulate furrow formation. However, some furrowing could be induced without aster formation in mature oocytes and unfertilized eggs by an activation stimulus, showing that asters are not essential for cleavage initiation. The significance of these observations are discussed in the light of our current understanding of meiotic maturation, cell cleavage and aster growth.

Animals↗
PubMed1977

Evidence for a functional role of RNA in centrioles.

Basal bodies, purified from Chlamydomonas and Tetrahymena, were exposed to various enzymatic treatments and then assayed for their ability to nucleate aster formation upon injection into eggs of Xenopus laevis. Untreated basal bodies injected into frog eggs act as centrioles and induce the formation of asters. The aster-inducing activity of basal bodies was eliminated by treatment with proteolytic enzymes and ribonucleases. Aster-inducing activity was not affected by DNAse and a number of other enzymes. The effect of proteolytic digestion on aster-inducing activity appeared to be directly correlated with the degree of structural damage to the basal body. Low concentrations of pancreatic ribonuclease A, ribonuclease T1, and S1 nuclease also completely abolished aster-inducing activity, although these enzymes had no effect on basal body structure. Ribonuclease-treated basal bodies remained capable of supporting microtubule elongation in vitro. Preliminary evidence indicates that basal bodies from Chlamydomonas and Tetrahymena contain about 5 x 10(-16) g of RNA which co-band with basal bodies and aster-inducing activity by equilibrium density gradient sedimentation. We conclude first, that centrioles contain RNA which is required for initiation of aster formation, and second, that the centriole activity or ability to assemble a mitotic aster is separable from the basal body activity, or ability to serve directly as a template for microtubule growth.

Animals↗
PubMed1975

Synthesis of soluble protein in oocytes of Xenopus laevis.

Oocytes of five stages of development were isolated from ovaries of Xenopus laevis and allowed to take up radioactive amino acids. After six hours of labeling the amount of label incorporated into perchloric acid precipitable material from the soluble oocyte fraction and the specific activity of the label free pool was determined. From these figures an estimate of the rate of protein synthesis was calculated. Labeled soluble protein from each oocyte stage was analyzed by electrophoresis on SDS polyacrylamide gels. The gel was dried and autoradiographed to determine the qualitative pattern of soluble protein synthesis during various stages of oogenesis. Our results indicate that no significant differences exist in the rate of protein synthesis among any of the stages of oogenesis investigated. The qualitative pattern of soluble protein synthesized during the labeling period is similar among the oocyte stages. Moreover, this qualitative pattern of soluble protein synthesis is the same as the pattern of soluble protein accumulated up to that time during oogenesis. These results suggest a stable synthesis and accumulation of maternal protein products during Xenopus oogenesis, in marked contrast to the results that have been reported for RNA synthesis during oogenesis in Xenopus. Our results are discussed in terms of the present understanding of the process of maternal information accumulation.

Animals↗
PubMed1975

Aster formation in eggs of Xenopus laevis. Induction by isolated basal bodies.

We have assayed various materials for their ability to induce aster formation by microinjection into unfertilized eggs of Xenopus laevis. We have found that purified basal bodies from Chlamydomonas reinhardtii and Tetrahymena pyriformis induce the formation of asters and irregular cleavage furrows within 1 h after injection. Other microtubule structures such as flagella, flagellar axonemes, cilia, and brain microtubules are completely ineffective at inducing asters or cleavage furrows in unfertilized eggs. When known amounts of sonicated Tetrahymena and Chlamydomonas preparations are injected into unfertilized eggs, 50% of the injected eggs show a furrowing response at approximately 3 cell equvalents for Chlamydomonas and 0.1 cell equivalent for Tetrahymena. These results are close to those expected if basal bodies were the effective astral-inducing agent in these cells. Other materials effective at inducing asters in unfertilized eggs, such as crude brain nuclei, sperm, and a particulate fraction from brain known to induce parthenogenesis in eggs of Rana pipiens, probably contain centrioles as the effective agent. Our experiments provide the first functional assay to indicate that centrioles play an active role in aster initiation. None of the injected materials effective in unfertilized eggs produced any observable response in fully grown oocytes. Oocytes and eggs were found to have equal tubulin pools as judged by colchicine-binding activity. Therefore, the inability of oocytes to form asters cannot be due to a lack of an organizing center or to a lack of tubulin. Experiments in which D2O was found to stimulate aster-like fibrous areas in eggs but not oocytes suggest that the inability of oocytes to form asters may be due to an inability of tubulin in oocytes to assemble.

Animals↗