The flagellar root system in the gamete ofAcetabularia mediterranea

Summary Ultrastructural investigation of the flagellar root system ofAcetabularia gametes reveals one type of organization for both male and female gametes. There is a modified cruciate system with four microtubular bands X-2-X-2, with X=4. A prominent distal striated fiber and a small proximal striated fiber connect the flagellar bases. A striated root fiber type I underlies the microtubular root type II, and a short striated root fiber type I underlies the microtubular root type I (terminology ofMelkonian 1980 b). This specific root system has some details in common with theChlamydomonas type, and others with theUlvaphyceae and the siphonalean algaeDerbesia andBryopsis. This might indicate the phylogenetic relationships.     

Division of the primary nucleus ofAcetabularia

Summary The division of the primary nucleus ofAcetabularia mediterranea, andAcetabularia cliftonii was studied by light microscopical observation of living cells. Nuclear and nucleolar volumes are reduced when the caps of the cells have reached their maximum diameter. When the nucleus has reached a size of about 30–50 m in diameter, condensed chromosomes are formed which are separated by an intranuclear spindle.     

The role of the osmotic potential of the cell in auxin-induced cell elongation

IAA-induced elongation of light-grown cucumber hypocotyl sectionswas examined with respect to the osmotic relationship of thecell. Sucrose suppression of IAA-induced elongation in the lightoccurred at a lower sucrose concentration than in the dark,but there was no difference in the mannitol concentration whichsuppressed elongation. This differential sucrose suppressioncould be explained by the difference in the osmotic potentialof the cells between light and darkness. It was lower in thedark than in light, and the difference was more distinct inthe presence of sucrose. Treatment of sections with a photosyntheticinhibitor, CMU, also resulted in the maintenance of a low osmoticpotential. Under the experimental conditions where a largerIAA-induced elongation was obtained, a lower osmotic potentialwas also obtained. The results are discussed with respect tothe role of the osmotic potential of the cell in the enhancementof IAAinduced elongation. (Received April 3, 1978; )     

The role of tubulin polymerization during spindle elongation in vitro

We describe the effect of exogenous tubulin on reactivation of anaphase spindle elongation in isolated diatom spindles. In the absence of tubulin, spindle elongation is limited to the equivalent of the microtubule overlap zone, but in the presence of tubulin spindle elongation is several times the length of the overlap zone. Biotinylated neurotubulin is incorporated into the overlap zone and around the poles. Before spindles have elongated by the equivalent of the overlap zone, there are two regions of incorporated tubulin flanking this zone. After further elongation, there is one broad zone of incorporated tubulin in the spindle midzone. Spindle elongation and the pattern of tubulin incorporation into the midzone, but not the poles, are ATP-dependent and vanadate-sensitive. These results suggest that tubulin adds onto the ends of microtubules in the overlap zone, which then slide through the midzone as the spindle elongates.     

The role of cholesteryl 14-methylhexadecanoate in peptide elongation reactions

1. Peptide-elongation factors were purified from rat liver and human tonsils and the contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during enzyme purification. The relative contents of this compound in purified enzyme preparations was several times higher than that in the crude starting material. Elongation factors from human tonsils contained a significantly larger quantity of the cholesteryl ester than enzyme from rat liver. 2. Transfer enzymes extracted with various organic solvents showed variable decreased activities in both binding and peptidization assay. The decrease of enzymic activity was proportional to the amount of cholesteryl 14-methylhexadecanoate extracted from a given enzymic preparation. In systems containing both extracted elongation factors the polyphenylalanine synthesis was limited by the residual activity of the less active transfer factor. 3. The original enzymic activity of extracted transferases was fully recovered by the addition of pure cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. Increase of the relative contents of this cholesteryl ester during enzyme purification, decrease of the enzymic activity after the extraction and its recovery by the addition of this compound indicates that the presence of this ester in elongation factors is essential for the normal function of these enzymes.     

Observations on the sterile whorls ofAcetabularia

Summary The sterile whorls ofAcetabularia increase greatly the surface area of the cell. These structures function in the uptake of solutes from the environment. The development of the whorls is controlled by light and also by the availability of nutrients.     

The role of the leaves in the regulation of internode elongation in Phaseolus vulgaris

Time-course patterns of leaf and internode elongation were studied in bean plants. Each leaf started its main elongation period when the leaf below reached half of its final length. The onset of leaf unfolding was nearly synchronous with the initiation of the elongation of the subjacent internode. Excision of young leaves increased the rate of stem elongation as a result of an earlier unfolding of the next upper leaves and the concomitant advancement in the elongation of their subjacent internodes. IAA or NAA (1% in lanolin) suppressed the enhancement effects of leaf excision on leaf and internode elongation. The excision of a young leaf increased the final length of internodes located below it, and at the same time decreased the final length of the internodes located above the excised leaf. The reduction was greater the younger the internode. Differences in internode elongation after leaf excision were related to changes during internode ontogenesis in their relative response to the availability of assimilates on the one hand, and on the other hand to hormonal factors transported acropetally from the young leaves to the growing internodes.     

Current-voltage relationships of potassium channels in the plasmalemma ofAcetabularia

Summary The outer membrane of mechanically prepared protoplasmic droplets fromAcetabularia mediterranea has been investigated by patch-clamp techniques. These membranes are shown to consist of physiologically intact plasmalemma. With the Cl^– pump inhibited, microscopic currents through K⁺-selective channels were studied. These currents compare well with macroscopic K⁺ currents as previously determined by standard microelectrode techniques and tracer flux measurements. There is about one K⁺ channel per m² in the plasmalemma. The current-voltage relationship (I–V curve) of the main open channel (channel A) is sigmoid over a voltage range between about –100 and +100 mV with saturation currents of about ±10 pA. A second species (or different state of channel A) of K⁺-selective channels (channel B) differs from channel A by smaller saturation currents (about ±7 pA) and a much smaller open probability. The open probability of channel A increases from almost zero at large negative voltages to about 1/2 at large positive voltages. Taking the closed times into account, the mean steady-stateI–V curve of channel A displays outward rectification about the equilibrium voltage for K⁺ and negative slope conductance at larger negative voltages. The open channelI–V curve of the open channels A and B, the changes of theI–V curve of the open channel A upon variation of the external K⁺ concentration, as well as the mean steady-stateI–V curves of channel A are described by simple reaction kinetic models, the parameters of which are determined to fit the experimental data. The results are discussed with respect to data from other K⁺ channels in plants and with respect to regulation of the cytoplasmic K⁺ concentration inAcetabularia.     

Ultrastructural features of the life cycle ofAcetabularia mediterranea

Summary The portion of the life cycle ofAcetabularia mediterranea from cyst formation to gamete release is described. During maturation, the cyst nucleus undergoes a series of mitoses in which the nuclear membrane remains intact, and the spindle microtubules are confined to the intranuclear space. There is a dark requirement for the completion of cyst maturation at the end of which the nuclei are in groups and centrioles are present. The final migration of the nuclei, cleavage of the cytoplasm into gametes, and the development of flagella is cxtremely rapid, taking six to twelve hours. Cysts which fail to germinate are blocked at a late stage in the maturation process, either before or after the final nuclear division.The observations are related to mitosis in other green algae (Chlorophyceae) and to gametogenesis inBryopsis hypnoides. Changes in the amount and distribution of heterochromatin in the nuclei are discussed in relation to the life cycle ofA. mediterranea.The substance of this paper was first presented orally at the Symposium onAcetabularia held at the Max-Planck-Institut für Zellbiologie, Wilhelmshaven, Federal Republic of Germany, July 12–15th, 1972.Supported in part by Grant GM 06637 from the U.S. Public Health Service.     

The role of light in suppressing hypocotyl elongation in lettuce and Petunia

Summary Hypocotyl elongation in two varieties of Petunia and in Grand Rapids lettuce is shown to be affected by a high-energy reaction and by phytochrome action. These two photoreactions interact in such a way that, on the one hand, shortening of the hypocotyls due to the high-energy reaction can be entirely masked by brief terminal far-red light treatment, while on the other hand, there is no evidence of phytochrome action unless brief exposures to red light are preceded by relatively long exposure of high-intensity.The action spectra for the high-energy reaction show peak effectiveness at wavelengths of 430–450 m, with a minor peak at 660 m in Comanche Petunia, at 700 m in Pink Cascade Petunia, and at 720 m in Grand Rapids lettuce.Prior treatment with DCMU did not reduce the effect of high-intensity light on hypocotyl lengths in lettuce.The nature of the high-energy reaction, and the relation between it and phytochrome action are discussed. Besides these two photoreactions there appears to be a direct effect of light on elongation, blue light preventing, and far-red light accelerating, elongation during actual exposure.With 9 Figures in the Text     

Multinuclear stages of the life cycle ofAcetabularia mediterranea

Summary The reproductive stages of the life cycle ofAcetabularia mediterranea have been studied in Feulgen-stained material. Light-microscopical photographs of secondary nuclei, cyst formation, gamete formation, gamete release, zygote formation and early development of the germlings are presented. The time course of cytological events during gamete formation is described. Mitoses are found between 16 and 24 hours after the induction of cyst germination.The author is indebted to Mrs. S.Artelt who helped with the prepration of the specimens and the photographs.     

Morphology of the nucleo-cytoplasmic interactions during the development ofAcetabularia cells

Protoplasma - An electron microscopic survey of nuclear events and changes in the perinuclear cytoplasm during the generative phase ofAcetabularia is presented with details on late stages in the...     

The role of cholesteryl 14-methylhexadecanoate in the function of eukaryotic peptide elongation factor 1

The binding of [3H]cholesteryl 14-methylhexadecanoate by a highly purified peptide elongation factor 1 from rabbit reticulocytes is significantly enhanced by GTP and CTP, much less by guanosine 5'-[beta, gamma-methylene]-triphosphate and not at all by ATP or UTP. Removal of endogenous cholesteryl 14-methylhexadecanoate present in the molecule of the factor [Hradec, J. et al. (1971) Biochem. J. 123, 959-966] by digestion with immobilized cholesterol esterase resulted in an almost complete loss of GTPase activity and this could be restored to nearly normal values by the addition of the ester. The same holds true for the GTP-dependent autophosphorylation of the protein-synthesis factor. Cholesteryl 14-methylhexadecanoate was bound only by the beta subunit of the factor. Addition of the alpha subunit, which was inactive on its own, stimulated the binding of the ester to the beta subunit in a sigmoid dependence. The binding of the ester was significantly stimulated by aminoacyl-tRNA but this effect was fully abolished by sodium fluoride, indicating a relation of cholesteryl 14-methylhexadecanoate to the dephosphorylation of the peptide elongation factor. Treatment of the factor with cholesterol esterase decreased its activity in the poly(U)-dependent binding of phenylalanyl-tRNA to ribosome and this activity was again restored by the addition of cholesteryl 14-methylhexadecanoate. The ester thus interacts with the GTP-dependent autophosphorylation of peptide elongation factor 1 and in this way modulates the activity of the factor. A putative scheme is presented explaining the mode of action of cholesteryl 14-methylhexadecanoate.     

The role of the epidermis in auxin-induced and fusicoccin-induced elongation of Pisum sativum stem segments

The effects of peeling and wounding on the indole-3-acetic acid (IAA) and fusicoccin (FC) growth response of etiolated Pisum sativum L. cv. Alaska stem tissue were examined. Over a 5 h growth period, peeling was found to virtually eliminate the IAA response, but about 30% of the FC response remained. In contrast, unpeeled segments wounded with six vertical slits exhibited significant responses to both IAA and FC, indicating that peeling does not act by damaging the tissue. Microscopy showed that the epidermis was removed intact and that the underlying tissue was essentially undamaged. Neither the addition of 2% sucrose to the incubation medium nor the use of a range of IAA concentrations down to 10^-8 M restored IAA-induced growth in peeled segments, suggesting that lack of osmotic solutes and supra-optimal uptake of IAA were not important factors over this time period. It is concluded that, although the possibility remains that peeling merely allows leakage of hydrogen ions into the medium, it seems more likely that peeling off the epidermis removes the auxin responsive tissue.Abbreviations IAA indole-3-acetic acid - FC fusicoccin     

The role of the FH1 domain and profilin in formin-mediated actin-filament elongation and nucleation

BACKGROUND: Formin proteins nucleate actin filaments de novo and stay associated with the growing barbed end. Whereas the formin-homology (FH) 2 domains mediate processive association, the FH1 domains-in concert with the actin-monomer-binding protein profilin-increase the rate of barbed-end elongation. The mechanism by which this effect is achieved is not well understood. RESULTS: We used total internal reflection fluorescence microscopy to measure the effect of profilin on the elongation of single actin filaments associated with FH1FH2 constructs (derived from the formin Bni1p from S. cerevisiae) with FH1 domains containing one to eight profilin-binding polyproline tracks. Over a large range of profilin concentrations (0.5-25 microM), the rate of barbed-end elongation increases with the number of polyproline tracks in the FH1 domain. The binding of profilin-actin to the FH1 domain is the rate-limiting step (up to rates of at least 88 s(-1)) in FH1-mediated transfer of actin subunits to the barbed end. Dissociation of formins from barbed ends growing in the presence of profilin is proportional to the elongation rate. Profilin profoundly inhibits nucleation by FH2 and FH1FH2 constructs, but profilin-actin bound to FH1 might contribute weakly to nucleation. CONCLUSIONS: To achieve fast elongation, formin FH1 domains bind profilin-actin complexes and deliver them rapidly to the barbed end associated with the FH2 domain. Because subunit addition promotes dissociation of FH2 domains from growing barbed ends, FH2 domains must pass through a state that is prone to dissociation during each cycle of actin subunit addition coupled to formin translocation.