MICROTUBULE BIOGENESIS AND CELL SHAPE IN OCHROMONAS : II. The Role of Nucleating Sites in Shape Development

The proposal made in the preceding paper that the species-specific shape of Ochromonas is mediated by cytoplasmic microtubules which are related to two nucleating sites has been experimentally verified. Exposure of cells to colchicine or hydrostatic pressure causes microtubule disassembly and a correlative loss of cell shape in a posterior to anterior direction. Upon removal of colchicine or release of pressure, cell shape regenerates and microtubules reappear, first in association with the kineto-beak site concomitant with regeneration of the anterior asymmetry, and later at the rhizoplast site concomitant with formation of the posterior tail. It is concluded that two separate sets of cytoplasmic tubules function in formation and maintenance of specific portions of the total cell shape. On the basis of the following observations, we further suggest that the beak and rhizoplast sites could exert control over the position and timing of the appearance, the orientation, and the pattern of microtubule distribution in Ochromonas. (a) the two sites are accurately positioned in the cell relative to other cell organelles; (b) in regenerating cells microtubules reform first at these sites and appear to elongate to the cell posterior; (c) microtubules initially reappear in the orientation characteristic of the fully differentiated cell; (d) the two sets of tubules are polymerized at different times, in the same sequence, during reassembly or resynthesis of the microtubular system. Experiments using cycloheximide, after a treatment with colchicine, have demonstrated that Ochromonas cannot reassume its normal shape without new protein synthesis. This suggests that microtubule protein once exposed to colchicine cannot be reassembled into microtubules. Pressure-treated cells, on the other hand, reassemble tubules and regenerate the normal shape in the presence or absence of cycloheximide. The use of these two agents in analyzing nucleating site function and the independent processes of synthesis and assembly of microtubules is discussed.     

MICROTUBULES IN THE FORMATION AND DEVELOPMENT OF THE PRIMARY MESENCHYME IN ARBACIA PUNCTULATA : I. The Distribution of Microtubules

Prior to gastrulation, the microtubules in the presumptive primary mesenchyme cells appear to diverge from points (satellites) in close association with the basal body of the cilium; from here most of the microtubules extend basally down the lateral margins of the cell. As these cells begin their migration into the blastocoel, they lose their cilia and adopt a spherical form. At the center of these newly formed mesenchyme cells is a centriole on which the microtubules directly converge and from which they radiate in all directions. Later these same cells develop slender pseudopodia containing large numbers of microtubules; the pseudopodia come into contact and fuse to form a "cable" of cytoplasm. Microtubules are now distributed parallel to the long axis of the cable and parallel to the stalks which connect the cell bodies of the mesenchyme cells to the cable. Microtubules are no longer connected to the centrioles in the cell bodies. On the basis of these observations we suggest that microtubules are a morphological expression of a framework which opeartes to shape cells. Since at each stage in the developmental sequence microtubules appear to originate (or insert) on different sites in the cytoplasm, the possibility is discussed that these sites may ultimately control the distribution of the microtubules and thus the developmental sequence of form changes.     

MICROTUBULE BIOGENESIS AND CELL SHAPE IN OCHROMONAS : I. The Distribution of Cytoplasmic and Mitotic Microtubules

In the first of two companion papers which attempt to correlate microtubules and their nucleating sites with developmental and cell division patterns in the unicellular flagellate, Ochromonas, the distribution of cytoplasmic and mitotic microtubules and various kinetosome-related fibers are detailed. Of the five kinetosome-related fibers, which have been found in Ochromonas, two, the kineto-beak fibers and the rhizoplast fibers are utilized as attachment sites for distinct groups of microtubules. The set of microtubules attached to the kineto-beak fibers apparently shape the anterior beak region of the cell whereas the rhizoplast microtubules appear to extend into and shape the tail in vegetative cells. In mitotic cells a rhizoplast is found at each spindle pole apparently serving as foci for the spindle microtubules. These findings are discussed in relation to the less well defined attachment sites for vegetative and mitotic microtubules in other kinds of cells. It is noted that the effects of depolymerizing microtubules in vivo might be easily quantitated in whole populations since no external wall or pellicle contributes to the maintenance or the biogenesis of the characteristic cell form of Ochromonas.     

Studies on the reactivity of malondialdehyde. I. Effects of acid concentration and solvent composition on deuterium exchange reactions

The rates of deuterium exchange reactions of malondialdehyde (MDA) and deuterated malondialdehyde (MDAd) have been studied as a function of acidity and the content of dimethyl sulfoxide (DMSO) in binary mixtures with D₂O . MDA incorporates deuterium from D₂O solutions in a first-order reaction with a rate constant (k_obs) that depends on the acid concentration. From this dependence, a catalytic constant, k_cat, can be derived (k_cat^MDA = 2.25 × 10⁵M^?s^?1). Similar kinetic behavior was found for MDAd in H₂O solutions, and in this case, k_cat^MDA = 1.56 × 10⁵M^?1s^?1. Results from reactions of MDA and MDAd in identical H₂OD₂O mixtures show that primary and secondary isotope effects are small (k_H/k_D = 1.13) and that solvent isotope effects cause most of the differences found between reactions in D₂O and H₂O. Reactions in binary DMSOd₆D₂O mixtures show a six-fold rate increase as the proportion of DMSOd₆ increases from 50% to 90%. These results also illustrate the relatively high reactivity of MDA at pH values well above its pK_a and the importance of medium composition on its reaction rate.     

Acid mucopolysaccharide metabolism, the cell surface, and primary mesenchyme cell activity in the sea urchin embryo

The relationship between ³⁵SO₄ incorporation into acid mucopolysaccharides and the appearance and activity of the primary mesenchyme cells has been studied in the sea urchin, Lytechinus pictus. The ratio of the uptake of ³⁵SO₄ to its incorporation into cetylpyridinium chloride precipitable material varies over a wide range during early development, with the smallest ratio, therefore the greatest sulfation activity, being found at the early mesenchyme blastula stage. The types of mucopolysaccharides produced have not been identified, but are heterogeneous. At the mesenchyme blastula stage nearly 90% of the polysaccharides produced become sulfated. When embryos develop in sulfate-free sea water to the mesenchyme blastula stage there is a 70% decrease in the incorporation of ³H-acetate into polysaccharides and a 13-fold decrease in the ratio of sulfated to nonsulfated polysaccharides produced. Embryos raised in sulfate-free sea water develop normally to the mesenchyme blastula stage at which time there is an accumulation in the blastocoel of primary mesenchyme cells that do not migrate. The surface of the primary mesenchyme cells of sulfate-deficient embryos has a smooth appearance in the scanning electron microscope, while the surface of these cells in control embryos is rough, possibly reflecting the presence of an extracellular coat. It is suggested that there is a correlation between sulfated polysaccharide synthesis, cell surface morphology and cell movement.     

Colchicine-binding protein and the secretion of thyroid hormone

The role of microtubules in the thyrotropin- or adenosine 3'',5'' cyclic monophosphate (cyclic AMP)-stimulated accumulation of cytoplasmic colloid droplets and secretion of iodine from the mouse thyroid gland has been investigated by means of different classes of agents that affect the stability of microtubules. The onset of inhibition of secretion by colchicine, the uptake of colchicine-³H by thyroid lobes, and the binding of colchicine-³H to thyroidal soluble protein are shown to have similar time courses Colloid droplet accumulation is also inhibited and does not readily resume upon removal of colchicine from the medium. This appears to be due to the slow washout of the drug (t _½ ∼ hr). Thyroids contain a soluble colchicine-binding protein that resembles microtubule proteins of other tissues with respect to apparent K_m for colchicine, pH optimum, and stability characteristics Colchicine analogues inhibit iodine secretion and colchicine binding in a parallel manner and as a function of their antimitotic potencies. Microtubule-stabilizing agents such as hexylene glycol and D₂O also inhibit secretion. Thus, inhibition of thyroid secretion by antimitotic agents appears to be mediated by an effect on microtubules. The inhibitory locus of colchicine inhibition occurs after the generation of cyclic AMP, since stimulation of secretion by this nucleotide is blocked by colchicine, whereas thyroid-stimulating hormone—induced accumulation of cyclic AMP is not affected. Thus, the functioning microtubule appears to play a role in the induction of colloid endocytosis.     

Solvent isotope effects on microtubule polymerization and depolymerization

The initial velocity of polymerization of purified beef brain tubulin has been determined at various values of pH or pD in water and in H₂O-D₂O mixtures. D₂O was shown to inhibit both polymerization at 37 °C and depolymerization measured at 5 °C and 37 °C. The microtubules formed in D₂O were indistinguishable from those formed in H₂O, by electron microscope examination. In 93% D₂O the pL²versus rate of polymerization curve was displaced about one unit towards higher pL values. In certain regions of the pL versus rate curve, a stimulation in the rate of polymerization by D₂O is observed. The extent of polymerization at the optimum pL value was not affected by D₂O.     

On the involvement of singlet oxygen in mutation induction by 8-methoxypsoralen and UVA irradiation in Escherichia coli K-12

The possible mutagenic effects induced by singlet oxygen, which is formed during UVA irradiation of bacterial cells pretreated with 8-methoxypsoralen (8-MOP), were investigated. As genetic endpoint, back mutation from arg₅₆^? to arg⁺ was assayed in strain Escherichia coli K-12/343/113/uvrB; this system, in preliminary experiments, was rather sensitive to 8-MOP-induced photodynamic effects. To assess the involvement of singlet oxygen (¹O₂) in the mutation induction process, 2 tests were applied, namely, comparative mutation induction in D₂O and in H₂O media (pH 7.0) and quenching of ¹O₂ with 1,4-diazabicyclo[2.2.2]octane (DABCO).When photodynamy was performed with the indicator cells suspended in D₂O buffer, the mutagenic effect was substantially higher than that obtained with cells suspended in H₂O buffer; this increase was even more pronounced when the incubation mixtures were thoroughly oxygenated before irradiation. D₂O itself was not mutagenic under the present experimental conditions. Addition of DABCO in concentrations of 0.1–10 mM to the irradiation mixtures effectively reduced the number of 8-MOP-induced mutant yields by about 40%. DABCO itself had no effect on cell viability or on spontaneous mutation frequency under our experimental conditions.From these 2 sets of results, and from the preliminary findings that the photomutagenic effect of 8-MOP is higher in the uvrB derivative than in the corresponding excision-repair-proficient parent strain, which is in concordance with previous observations in other E coli strains, it can be concluded that ¹O₂ generated upon UVA irradiation of 8-MOP solutions is probably responsible for part of the observed genetic effects.     

Nuclear Magnetic Resonance Evidence using D2O for Structured Water in Muscle and Brain

The electric quadrupole moment of the deuterium nucleus provides a nuclear magnetic resonance (NMR) probe of electric field gradients, and thereby of organization of tissue water. 8-17% of H₂O in rat muscle and brain was replaced by D₂O from 50% deuterated drinking water. The peak height of the steady-state NMR spectrum of D in muscle water was 74% lower than that of an equal concentration of D₂O in liquid water. Longitudinal NMR relaxation times (T₁) of D in water of muscle and brain averaged 0.092 and 0.131 sec, respectively, compared with 0.47 sec in D₂O in liquid water. Transverse NMR relaxation times (T₂) averaged 0.009 and 0.022 sec in D₂O of muscle and brain, respectively, compared with 0.45 sec in D₂O in liquid water. These differences cannot be explained by paramagnetic ions or by magnetic inhomogeneities, which leaves increased organization of tissue water as the only tenable hypothesis. Evidence was also obtained that 27% of muscle water and 13% of brain water exist as a separate fraction with T₂ of D₂O less than 2 × 10^-3 sec, which implies an even higher degree of structure. Each of the two fractions may consist of multiple subfractions of differing structure.     

1,2H hyperfine spectroscopy and DFT modeling unveil the demethylmenasemiquinone binding mode to E. coli nitrate reductase A (NarGHI)

The quinol oxidation site Q_D in E. coli respiratory nitrate reductase A (EcNarGHI) reacts with the three isoprenoid quinones naturally synthesized by the bacterium, i.e. ubiquinones (UQ), menaquinones (MK) and demethylmenaquinones (DMK). The binding mode of the demethylmenasemiquinone (DMSK) intermediate to the EcNarGHI Q_D quinol oxidation site is analyzed in detail using ^1,2H hyperfine (hf) spectroscopy in combination with H₂O/D₂O exchange experiments and DFT modeling, and compared to the menasemiquinone one bound to the Q_D site (MSK_D) previously studied by us. DMSK_D and MSK_D are shown to bind in a similar and strongly asymmetric manner through a short (~1.7 Å) H-bond. The origin of the specific hf pattern resolved on the DMSK_D field-swept EPR spectrum is unambiguously ascribed to slightly inequivalent contributions from two β-methylene protons of the isoprenoid side chain. DFT calculations show that their large isotropic hf coupling constants (A_iso ~12 and 15 MHz) are consistent with both (i) a specific highly asymmetric binding mode of DMSK_D and (ii) a near in-plane orientation of its isoprenyl chain at Cβ relative to the aromatic ring, which differs by ~90° to that predicted for free or NarGHI-bound MSK. Our results provide new insights into how the conformation and the redox properties of different natural quinones are selectively fine-tuned by the protein environment at a single Q site. Such a fine-tuning most likely contributes to render NarGHI as an efficient and flexible respiratory enzyme to be used upon rapid variations of the Q-pool content.     

Expression of CXCL12 and CXCL14 during eye development in chick and mouse

Vertebrate eye development is a complex multistep process coordinated by signals from the lens, optic cup and periocular mesenchyme. Although chemokines are increasingly being recognized as key players in cell migration, proliferation, and differentiation during embryonic development, their potential role during eye development has not been examined. In this study, we demonstrate by section in situ hybridization that CXCL12 and CXCL14 are expressed during ocular development. CXCL12 is expressed in the periocular mesenchyme, ocular blood vessels, retina, and eyelid mesenchyme, and its expression pattern is conserved between chick and mouse in most tissues. Expression of CXCL14 is localized in the ocular ectoderm, limbal epithelium, scleral papillae, eyelid mesenchyme, corneal keratocytes, hair follicles, and retina, and it was only conserved in the upper eyelid ectoderm of chick and mouse. The unique and non-overlapping patterns of CXCL12 and CXCL14 expression in ocular tissues suggest that these two chemokines may interact and have important functions in cell proliferation, differentiation and migration during eye development.     

Effect of deuterium oxide on contraction characteristics and ATPase activity in glycerinated single rabbit skeletal muscle fibers

We studied the effect of deuterium oxide (D₂O) on contraction characteristics and ATPase activity of single glycerinated muscle fibers of rabbit psoas. D₂O increased the maximum isometric force P₀ by about 20%, while the force versus stiffness relation did not change appreciably. The maximum shortening velocity under zero load V_max did not change appreciably in D₂O, so that the force-velocity (P-V) curve was scaled depending on the value of P₀. The Mg-ATPase activity of the fibers during generation of steady isometric force P₀ was reduced by about 50% in D₂O. Based on the Huxley contraction model, these results can be accounted for in terms of D₂O-induced changes in the rate constants f₁ and g₁ for making and breaking actin-myosin linkages in the isometric condition, in such a way that f₁/(f₁+g₁) increases by about 20%, while (f₁+g₁) remains unchanged. The D₂O effect at the molecular level is discussed in connection with biochemical studies on actomyosin ATPase.     

STUDIES ON THE MICROTUBULES IN HELIOZOA : V. Factors Controlling the Organization of Microtubules in the Axonemal Pattern in Echinosphaerium (Actinosphaerium) nucleofilum

On the assumption that the double-coiled pattern of microtubules in the axoneme of Echinosphaerium might be due to links of two sizes between adjacent microtubules, we disassembled microtubules with low temperature and then carefully analyzed the patterns of microtubules that formed upon the addition of heat (22°C) or heat and D₂O. Although most of the initial clusters of microtubules that formed could not be interpreted as part of an axoneme, the spacings between these microtubules were the same as that in the axoneme, 70 and 300 A. By model building we were able to show that all clusters that form, including stages in the formation of the axoneme and its 12-fold symmetry, could be explained by links of two sizes (70 and 300 A) and the substructure of the microtubule. We could demonstrate these links with improved staining methods. We suggest that nonaxonemal assemblies of microtubules may be eliminated by the natural selection of the most energetically stable configuration of microtubules, all others undergoing disassembly under equilibrium conditions. Model building further supports this suggestion since the model axoneme possesses more links per tubule than any other cluster found.     

Effect of temperature on the embryonic development of the plaice, Pleuronectes Platessa L. (teleostei)

In normalyears, eggs and prolarvae of the plaice (Pleuronectes platessa L.) in the southern North Sea develop within the temperature range 6.0–8.5 °C, although the water may at times be some degrees colder or warmer than this. The effect of temperature, t °C, on the embryonic development time, D days, has been investigated within the tolerated range 2.8–10.5 °C. Various models to express the observed curvilinear relationship between t and D have been considered, that giving the closest fit to the data being (t−t₀)(D−D₀) = k or $\text{D =}\text{k}\text{(t−t}{}_0\text{)+D}{}_0$. A method is given for the calculation of constants k, D₀, and t₀. The relationship may also be expressed by the equation D = a(t−t₀)^b where a and b are constants, but t₀ must in this case be found by iteration. At investigated temperatures in the range 4.1–10.5 °C the smallest eggs in a batch from a single source hatched first. Within the tolerated range, hatching prolarvae were substantially smaller at 10.5 °C than at the other temperatures. During the period of prolarval yolk utilization, growth is slower at the high temperatures, so that median temperatures of 6.5–8.0 °C are most efficient in terms of the relationship between growth in length and yolk utilization. Toward the end of the yolk-sac phase, the rate of yolk utilization declines unless a suitable external food source (e.g., Artemia nauplii) is provided.     

The program of protein synthesis during the development of the micromere-primary mesenchyme cell line in the sea urchin embryo

Changes in the pattern of protein synthesis were analyzed during the in vitro development of the micromere-primary mesenchyme cell line of the sea urchin embryo. Micromeres were isolated and cultured from 16-cell stage embryos, and primary mesenchyme cells were isolated and cultured from early gastrulae. Both cell isolates developed normally in culture with about the same timing as their in situ counterparts in control embryos. Newly synthesized proteins were labeled with [³H]valine at several stages of development and were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorgraphy. The electrophoretic pattern of labeled proteins changed dramatically during development. More than half of the analyzed proteins underwent qualitative or quantitative changes in their relative rates of valine incorporation and these changes were highly specific to this cell line. Almost all of the changes were initiated prior to gastrulation and many prior to hatching. The highest frequency of changes in the micromere pattern of protein synthesis occurred between hatching and the start of gastrulation. This peak of activity coincided with the normal time of ingression of the primary mesenchyme and preceded the differentiation of spicules by more than 30 hr. Most of the observed changes were characterized as either decreases in the synthesis of proteins that showed maximum incorporation at the 16-cell stage or increases in the synthesis of proteins that showed maxima in the fully differentiated cells. Very few proteins exhibited transient synthetic maxima at intermediate stages. Thus, the program of protein synthesis associated with the development of micromeres consists largely of a switch in emphasis from early to late proteins, with the primary time of switching being between hatching and the onset of gastrulation.     

Incipient Genome Differentiation in Gossypium. I. Chromosomes 14, 15, 16, 19 and 20 Assessed in G. HIRSUTUM, G. RAIMONDII and G. LOBATUM by Means of Seven a-D Translocations

The genus Gossypium is favorable for study of genome divergence at several levels. Early stages of divergence have been studied among four D genomes by comparing chiasma frequencies (reciprocal exchanges) between pairs of genomes and between individual counterpart chromosomes marked by heterozygous translocations. D₅ (G. raimondii) shows barely detectable differentiation from from D_h (G. hirsutum), whereas D₇ (G. lobatum) is considerably less closely related to D_h than is D₅. Fragmentary data suggest that D_2–2 (G. harknessii) falls between D₅ and D₇ in its relationship to D_h. Since chiasma frequencies in individual chromosomes and marked regions exhibit the same order of relationships as their corresponding whole genomes, it is concluded that the genome differentiation is generalized (i.e., nucleus-wide) rather than localized in specific chromosomes or chromosome regions. Estimates of relationships based on reciprocal exchange frequencies agree with those based upon preferential synapsis in allohexaploids reported previously. Since preferential synapsis and reciprocal exchange frequencies reveal the same order of relationships, it is concluded that to some extent they reflect common underlying changes in chromosome properties, despite recent evidence that synapsis and crossing over are under independent genetic control.     

Purification, properties, and kinetic observations on the isoenzymes of NADP isocitrate dehydrogenase of maize.

A successful method for the purification of NADP isocitrate dehydrogenase from a plant source, Zea mays, is reported. Two mitochondrial isoenzymes were found and purified to homogeneity by a course of acetone fractionation, bulk exchange on DEAE-cellulose, cellulose hydroxylapatite column chromatography, and continuous elution electrophoresis. The mitochondrial isoenzymes are very similar with respect to kinetic properties, response to solvent perturbation, and temperature dependence of the pH/V relationship of isocitrate dehydrogenation. The Michaelis constant for isocitrate is identical for both isoenzymes. The enzymes have a molecular weight of 81,000 as estimated by permeation chromatography and an isoelectric point of 5.5 as extrapolated from gel-electrophoretic mobilities. Detectable differences are confined to differences in electrophoretic mobilities and heat denaturation. In D₂O the rate of the overall reaction from isocitrate to α-ketoglutarate and CO₂ was about 3.6 times slower than the same reaction in H₂O. Both the forward and reverse reactions, in which isocitrate is dehydrogenated or generated from oxalosuccinate, were observed to decrease by this amount in D₂O. The decarboxylation of oxalosuccinate was found to decrease by only about 25% in D₂O relative to the velocity of the reaction in H₂O. Thus the slow step in the overall reaction must be the initial dehydrogenation step rather than the decarboxylation of oxalosuccinate. The pK of the overall reaction did not change in D₂O as compared to H₂O.