Polyphosphoinositol lipids inChlamydomonas eugametos gametes

InChlamydomonas eugametos gametes, phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) comprised 0.4 and 0.3% of the whole-cell phospholipids. They were concentrated in the plasma membrane around the cell body and were present in low concentrations in the flagellar membrane. When gametes were fed³²PO ₄ ^- , the label was rapidly incorporated into PtdInsP and PtdInsP₂ and only slowly incorporated into structural lipids such as phosphatidylethanolamine and phosphatidylglycerol. Similarly, when a pulse of³²PO ₄ ^- was chased with PO ₄ ^- , the label was rapidly lost from the polyphosphoinositol lipids but not from the structural lipids. The major fatty acids in the polyphosphoinositides were C-22 carbon polyenoic acids (70%). The significance of these results in relationship to intracellular signalling via inositol phosphates and Ca²⁺ is discussed.Abbreviations InsP₃ inositol 1,4,5-trisphosphate - mt^–/mt⁺ mating-type plus or minus - PtdA phosphatidic acid - PtdEtn phosphatidylethanolamine - PtdGro phosphatidylglycerol - PtdIns phosphatidylinositol - PtdInsP phosphatidylinositol 4-phosphate; - PtdInsP₂ phosphatidylinositol 4,5-bisphosphate - TCA trichloroacetic acid We thank Frank Schuring for Fig. 5A and Susan Kenter, Hans Kruisselbrink, Saskia Bijvank and Nelleke Corbett for their enthousiastic assistance.     

Polyphosphoinositide phospholipase C and evidence for inositol-phosphate-hydrolysing activities in the plasma-membrane fraction from light-grown wheat (Triticum aestivum L.) leaves

The phospholipase C (PLC; EC 3.1.4.3) activity in isolated plasma membranes of light-grown wheat (Triticum aestivum L. cv. Prelude) leaves was investigated. The activity against the polyphosphoinositides was strongly dependent on Ca²⁺ and was affected by the anionic detergent deoxycholate (DOC). In the presence of 20 M Ca²⁺ the PLC activity preferred phosphatidylinositol 4,5-bisphosphate (PIP₂) over phosphatidylinositol 4-monophosphate (PIP) as a substrate. Instead, with 1 mM Ca²⁺ the enzyme clearly favoured PIP. In addition, the PIP₂-PLC activity was increased by Mg²⁺ and in the presence of GTP, guanosine 5-(-thio)-triphosphate as well as ATP, CTP, guanosine 5-diphosphate and guanosine 5-(-thio)-diphosphate. Further analysis showed that a molybdate-sensitive phosphatase activity catalysing the dephosphorylation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) is also associated with the plasma-membrane vesicles. Dephosphorylation of Ins(1,4,5)P₃ was reduced in the presence of GTP or by inclusion of the unspecific phosphatase inhibitor molybdate. The results indicate the presence of a PIP₂-PLC activity and the presence of a molybdate-sensitive phosphatase activity in wheat plasma-membrane vesicles.Abbreviations DOC deoxycholate - IDPase inosine 5-diphosphatase - InsP_s inositol phosphates, the numbering at the end indicates the number of phosphate residues and when their positions on the inositol ring are known they are indicated in parentheses, i.e. - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PIP phosphatidylinositol 4-monophosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PLC phospholipase C This work was financially supported by grant from the Deutsche Forschungsgemeinschaft (DFG). M. C. Arz gratefully acknowledges the support of a Graduiertenstipendium des Landes Nordrhein-Westfalen (Germany). We wish to thank S. Laden and G.E. Grambow for assistance.     

Ethanol stimulates phospholipid turnover and inositol 1,4,5-trisphosphate production in Chlamydomonas eugametos gametes

Alcohols induce mating-structure activation in Chlamydomonas eugametos gametes. From the effect of ethanol on the ³²P-labelling of polyphosphoinositides, we conclude that the synthesis of these lipids is stimulated. Biologically inactive concentrations of ethanol (<6%) had no effect on synthesis, but 6–8% ethanol stimulated synthesis for upto 60 min. The ³²P incorporated into polyphosphoinositides and phosphatidic acid during ethanol treatment was readily chased out when 1 mM unlabelled Na₃PO₄ was added. Using a binding assay for inositol 1,4,5-trisphosphate, we show that the production of this phospholipid constituent is dramatically increased after ethanol treatment. This effect, coupled to a rise in intracellular calcium concentration, could explain gamete activation. The significance of these results in explaining other ethanol-induced phenomena in algae is discussed.Abbreviations Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PtdA phosphatidic acid - PtdIns phosphatidylinositol - PtdIns(4)P phosphatidylinositol 4-phosphate - PtdIns(4,5)P₂ phosphatidylinositol 4,5-bisphosphate To whom correspondence should be addressedWe thank Dr. P. van Haastert (Biochemistry, University of Groningen, The Netherlands) and his colleagues for introducing us to their Ins(1,4,5)P₃ assay, and Ben ten Brink (Molecular Cell Biology, University of Amsterdam, The Netherlands) for information about contractile vacuoles. We also thank Bas Nagelkerken, Marcel van der Vaart, Pieter van der Schoor, Gyuri Fenyvesi and Susan Kenter for their help.     

Inositol 1,4,5-trisphosphate may mediate closure of K+ channels by light and darkness in Samanea saman motor cells

Leaflet movements of Samanea saman (Jacq.) Merr. depend in part upon circadian-rhythmic, light-regulated K⁺ fluxes across the plasma membranes of extensor and flexor cells in opposing regions of the leaf-moving organ, the pulvinus. We previously showed that blue light appears to close open K⁺ channels in flexor protoplasts during the dark period (subjective night) (Kim et al., 1992, Plant Physiol 99: 1532–1539). In contrast, transfer to darkness apparently closes open K⁺ channels in extensor protoplasts during the light period (subjective day) (Kim et al., 1993, Science 260: 960–962). We now report that both these channel-closing stimuli increase inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] levels in the appropriate protoplasts. If extensor cells are given a pulse of red light followed by transfer to darkness, channels still apparently close (Kim et al. 1993) but changes in Ins(1,4,5)P₃ levels are complex with an initial decrease under red light followed by accumulation. Neomycin, an inhibitor of polyphosphoinositide hydrolysis, inhibits both blue-light-induced Ins(1,4,5)P₃ production and K⁺-channel closure in flexor protoplasts and both dark-induced Ins(1,4,5)P₃ production and K⁺ channel closure in extensor protoplasts. The G-protein activator, mastoparan, mimics blue light and darkness in that it both increases Ins(1,4,5)P₃ levels and closes K⁺ channels in the appropriate cell type at the appropriate time. These results indicate that phospholipase C-catalyzed hydrolysis of phosphoinositides, possibly activated by a G protein, is an early step in the signal-transduction pathway by which blue light and darkness close K⁺ channels in S. saman pulvinar cells.Abbreviations DiS-C₃-(5) 3,3-dipropylthiadicarbocyanine iodide - F measure change in Dis-C₃-(5) fluorescence - F_o initial Dis-C₃-(5) fluorescence - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PtdIns(4,5)P₂ phosphatidylinositol 4,5-bisphosphate - rbc red blood cell Supported by grants from NSF (IBN 9206179 and MCB 9305154) and U.S.-Israel Binational Agricultural Research and Development Fund (IS-1670-90RC) to R.C.C. We thank the University of Connecticut Biotechnology Center for the use of a fluorescent spectrophotometer.     

Sex-specific binding and inactivation of agglutination factor in Chlamydomonas eugametos

Gametes of opposite mating type (mt ⁺ and mt ^-) of the green alga Chlamydomonas eugametos agglutinate via their flagella as a prelude to sexual fusion. To quantitate sexual agglutination, an in vitro assay has been developed using ³⁵S-labeled flagella and the isolated mt ^-agglutination factor. It is shown that not only isolated flagella, but also the mt ^-agglutination factor rapidly bind to the flagella of intact gametes of the opposite mating type. This confirms the role of the mt ^-agglutination factor in determining the sexual agglutinability of mt ^-gametes. As a function of binding, the agglutinative power of the flagella of both mating types is destroyed by a temperature-sensitive process. Likewise, the mt ^-agglutination factor can be completely inactivated.Abbreviations Mt ^+/- mating type plus or minus - PAS periodic-acid Schiff-reagent - Hepes 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid - HMC buffer Hepes buffer (10 mM. pH 7.2, containing 1 mM MgCl₂ and 1 mM CaCl₂)     

Ultrastructure and properties of the sexual agglutinins of the biflagellate green alga Chlamydomonas moewusii

Summary The mt^– agglutinins of the interfertile species Chlamydomonas moewusii and Chlamydomonas eugametos are very similar fibrous molecules. The mt^– agglutinin of C. moewusii has the same Stokes radius (39 nm) and sedimentation coefficient (9.3 S) as its counterpart in C. eugametos; its length (336 nm) and its ultrastructure, including the position of four kinks are also the same as in C. eugametos. The sugar compositions of both agglutinins are very similar, and they react equally well with the monoclonal antibody Mab 66.3 raised against the mt^– agglutinin of C. eugametos. Finally, they are equally thermoresistant, with half-lives at 100 °C of 50 min (C. moewusii) and 57 min (C. eugametos). The mt⁺ agglutinins of both species are different. Both are fibrous molecules with a terminal head, but the fibrous part of the molecule in C. moewusii is shorter (210 nm compared to 276 nm). The mt⁺ agglutinin of C. moewusii is also significantly more sensitive to heating with a half-life of 6 min at 40 °C compared to the 20 min shown by the mt⁺ agglutinin of C. eugametos. Their sugar compositions are, however, very similar, and they react equally well with Mab 66.3. The mt⁺ agglutinin of C. moewusii is sensitive to denaturing reagents and proteolytic attack, whereas the mt^– agglutinin is highly resistant. It is proposed that the globular head of the mt⁺ agglutinin acts as its recognition domain and interacts with a carbohydrate ligand on the mt^– agglutinin.     

Sexual agglutination in Chlamydomonas eugametos before and after cell fusion

A new study of sexual agglutination between Chlamydomonas eugametos gametes and between vis-à-vis pairs has been made using techniques that allow one to distinguish between the flagella or cell bodies of individual mating types (mt⁺ or mt^-). It is shown that before mt⁺ and mt^- gametes fuse in pairs, their flagella, which adhere over their whole length, are maintained in a particular conformation around the mt^- cell body. In clumps of agglutinating gametes the cells are asymmetrically distributed with the mt⁺ gametes constituting the outer surface of the clumps with the mt^- gametes on the inside. The flagella are then all directed towards the middle of the clump. This orientation of the flagella is maintained for approx. 8 min after cell fusion before the vis-à-vis pair becomes motile. At this stage, all the flagellar tips are activated. The original mt⁺ flagellar tips then deactivate and swimming is resumed. The original mt^- flagella remain immotile and activated after cell fusion and eventually shorten by a third, but only 30 min or more after fusion. Motile vis-à-vis pairs eventually settle to the substrate when the gamete bodies fuse completely to form a zygote. Settling vis-à-vis pairs are attracted to those that have already settled, to glutaraldehyde-fixed pairs and to flagella isolated from mt^- gametes. They are not chemotactically attracted, rather they are weakly agglutinated. Living vis-à-vis pairs can be shown to aggregate in rows with the cell bodies lying side by side. It is argued that the flagellar agglutination sites involved in gamete recognition are also involved in vis-à-vis pair aggregationAbbreviations mt^+/- mating type plus or minus - FTA flagellar tip activation     

An inhibitor of cell fusion in Chlamydomonas eugametos

By a short treatment with acid of mt ^- gametes of Chlamydomonas eugametos, a factor is released which prevents gametic cell fusion, without affecting the viability of the cells. It has a very rapid action. By means of scanning electron microscopy it is shown that the factor has no influence on flagellar adhesion nor on the formation of a plasma papilla by cells of either mating type, but that it specifically inhibits the fusion of these papillae. Evidence is presented suggesting that this inhibitor has a predominant effect on mt ⁺ gametes. In cell pairs which are blocked with respect to papillar fusion, no flagellar disengagement occurs, which indicates that loss of agglutinability is a direct consequence of cell fusion.     

The substrate specificity of phosphoinositide phospholipase C in rat heart sarcolemma

In rat cardiac sarcolemmal membranes a phosphoinositide-specific phospholipase C (PLC) was found to be present. The enzyme hydrolysed exogenous [³H-]phosphatidylinositol 4,5-biphosphate ([³H-]PtdIns(4,5)P ₂) in an optimized assay mixture containing 15 leg SL protein, 100 mM NaCl, 1 mM free Ca²⁺,14 mM Na-cholate and 20 AM [³H-]PtdIns (4,5)P ₂ (400–500 dpm/gm-l) in 30 mM HEPES-Tris buffer (pH 7.0). The average specific activity was 9.14±0.55 nmol-mg^–1·2.5 min^–1. The addition of Mg²⁺ to the assay mixture did not change PLC activity but increased the relative amounts of dephosphorylated inositol products. In the absence of Na⁺ and at a low Ca²⁺ concentration (0.3 M), Mg²⁺ also enhanced the intraSL levels of PtdIns4P and PtdIns, and, moreover, inhibited PLC activity (IC₅₀0.07 mM). PtdIns4P seemd to be a good substrate for the rat SL PLC (23.07 ± 1.57 nmol·mg^–1·2.5 min^–1) whereas PtdIns was hydrolysed at a very low rate (0.36 ± 0.08 nmol·mg^–1·2.5 min^–1). Unlike PtdIns(4,5)P ₂, PLC-dependent PtdIns4P and PtdIns hydrolysis was not inhibited by Ca²⁺ concentrations over 1 mM. The possibility of distinct isozymes being responsible for the different hydrolytic activities is discussed. (Mol Cell Biochem116: 27–31, 1992).Abbreviations DAG sn-1,2-diacylglycerol - EGTA ethyleneglycol-O,O-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - Ins(1,4,5)P ₃ inositol 1,4,5-trisphosphate - InsP inositol monophosphate (unidentified isomer) - InsP ₂ inositol bisphosphate (unidentified isomer) - InsP ₃ inositol trisphosphate (unidentified isomer) - InsP _x any inositol phosphate - PLC phospholipase C - PtdIns phosphatidylinositol - PtdIns(4,5)P ₂ phosphatidylinositol 4,5-bisphosphate - PtdIns4P phosphatidylinositol 4-monophosphate - SL sarcolemma     

Inositol 1,4,5-trisphosphate 3-kinase activity in frog skeletal muscle

Frog skeletal muscle contains a kinase activity that phosphorylates inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. The inositol 1,4,5-trisphosphate 3-kinase activity was mainly recovered in the soluble fraction, where it presented a marked dependency on free calcium concentration in the physiological range in the presence of endogenous calmodulin. At pCa 5, where the activity was highest, the soluble 3-kinase activity displayed a K_m for inositol 1,4,5-trisphosphate of 1.6 μM and a V_max value of 25.1 pmol mg⁻¹ min⁻¹. The removal rates of inositol 1,4,5-trisphosphate by 3-kinase and 5-phosphatase activities of the total homogenate under physiological ionic conditions were very similar, suggesting that both routes are equally important in metabolizing inositol 1,4,5-trisphosphate in frog skeletal muscle.     

Rapid accumulation and metabolism of polyphosphoinositol and its possible role in phytoalexin biosynthesis in yeast elicitor-treated<Emphasis Type="Italic"> Cupressus lusitanica</Emphasis> cell cultures

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] rapidly accumulates in elicited Cupressus lusitanica Mill. cultured cells by 4- to 5-fold over the control, and then it is metabolized. Correspondingly, phospholipase C (PLC) activity toward phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] is stimulated to high levels by the elicitor and then decreases whereas Ins(1,4,5)P₃ phosphatase activity declines at the beginning of elicitation and increases later. These observations indicate that elicitor-induced biosynthesis and dephosphorylation of Ins(1,4,5)P₃ occur simultaneously and that the Ins(1,4,5)P₃ level may be regulated by both PtdIns(4,5)P₂–PLC and Ins(1,4,5)P₃ phosphatases. Studies on the properties of PLC and Ins(1,4,5)P₃ phosphatases indicate that PLC activity toward PtdIns(4,5)P₂ was optimal at a lower Ca²⁺ concentration than activity toward phosphatidylinositol whereas Ins(1,4,5)P₃ phosphatase activity is inhibited by high Ca²⁺ concentration. This suggests that Ins(1,4,5)P₃ biosynthesis and degradation may be regulated by free cytosolic Ca²⁺. In addition, a relationship between Ins(1,4,5)P₃ signaling and accumulation of a phytoalexin (-thujaplicin) is suggested because inhibition or promotion of Ins(1,4,5)P₃ accumulation by neomycin or LiCl affects elicitor-induced production of -thujaplicin. Moreover, ruthenium red inhibits elicitor-induced accumulation of -thujaplicin while thapsigargin alone induces -thujaplicin accumulation. These results suggest that Ca²⁺ released from intracellular calcium stores may mediate elicitor-induced accumulation of -thujaplicin via an Ins(1,4,5)P₃ signaling pathway, since it is widely accepted that Ins(1,4,5)P₃ can mobilize Ca²⁺ from intracellular stores. This work demonstrates an elicitor-triggered Ins(1,4,5)P₃ turnover, defines its enzymatic basis and regulation, and suggests a role for Ins(1,4,5)P₃ in elicitor-induced phytoalexin accumulation via a Ca²⁺ signaling pathway.Abbreviations Ins(1,4,5)P₃ Inositol-1,4,5-trisphosphate - Ins(1,4)P₂ Inositol-1,4-bisphosphate - Ins(4,5)P₂ Inositol-4,5-bisphosphate - Ins(1)P Inositol 1-phosphate - Ins(4)P Inositol 4-phosphate - PLC Phospholipase C - PtdIns Phosphatidylinositol - PtdIns(4,5)P₂ Phosphatidylinositol 4,5-bisphosphate - YE Yeast elicitor     

A rapid attenuation of muscarinic agonist stimulated phosphoinositide hydrolysis precedes receptor sequestration in human SH-SY-5Y neuroblastoma cells

Agonist occupancy of muscarinic cholinergic receptors in human SH-SY-5Y neuroblastoma cells elicited two kinetically distinct phases of phosphoinositide hydrolysis when monitored by either an increased mass of inositol 1,4,5-trisphosphate, or the accumulation of a total inositol phosphate fraction. Within 5s of the addition of the muscarinic agonist, oxotremorine-M, the phosphoinositide pool was hydrolyzed at a maximal rate of 9.5%/min. This initial phase of phosphoinositide hydrolysis was short-lived (t_1/2=14s) and after 60s of agonist exposure, the rate of inositol lipid breakdown had declined to a steady state level of 3.4%/min which was then maintained for at least 5–10 min. This rapid, but partial, attenuation of muscarinic receptor stimulated phosphoinositide hydrolysis occurred prior to the agonist-induced internalization of muscarinic receptors.Abbreviations I(1,4,5)P₃ inositol 1,4,5-trisphosphate - IP total inositol phosphate fraction - IPL total inositol lipid fraction - mAChR muscarinic acetylcholine receptor - NMS N-methylscopolamine - Oxo-M oxotremorine-M - PI phosphatidylinositol - PIP phosphatidylinositol 4-phosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PPI phosphoinositide - QNB quinuclidinyl benzilate Special issue dedicated to Dr. Bernard W. Agranoff     

Regulation of aplanospore germination in Vaucheria

Germination of aplanospores in Vaucheria longicaulis Hoppaugh var. macounii Blum proceeds through three stages of development. Stage I begins with the initiation of germination and lasts approx. 2 h. During this stage germinating filaments grow at an accelerated rate (266 ± 12 m · h^–1). Stage II is characterized by a sharp decline in the growth rate of germinating filaments (96 ± 4 m · h^–1) and lasts 4 h. This is followed, during the next 4 h, by a recovery in the growth rate (168 ± 8 m · h^–1) of germinating filaments, stage III. Growth rates stabilize and remain unchanged during subsequent development (Oliveira and Fitch, 1988, J. Submicrosc. Cytol. Pathol. 20, 397–406). The Ca²⁺-influx modulators LaCl₃, nifedipine and methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4 (2-trifluoromethylphenyl)-pyridine-5-carboxylate (Bay K-8644), the ionophore calcimycin (A23187), the intracellular Ca²⁺-release antagonist 8-N-N'-(diethylamino)-octyl-3,4, 5-trimethoxybenzoate (TMB-8), the Ca²⁺-uptake inhibitor ruthenium red and the phosphoinositide-cycle modulators LiCl and myo-inositol show that the events required for the initiation are distinct from those required for the completion of each stage of germination. These studies in conjunction with microinjection of germinating filaments with inositol 1,4,5-trisphosphate, the natural ligand for Ca²⁺ release from Ca-storing organelles (endoplasmic reticulum, vacuole), and treatment with chlorotetracycline (CTC), to visualize the distribution of membrane-bound Ca²⁺ reveal that both the initiation and completion of each stage of germination are controlled by Ca²⁺ signals which are restricted to well-defined time intervals and are modulated by the origin (source) of Ca²⁺.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - Bay K-8644 methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4(2-trifluoromethylphenyl)-pyridine-5-carboxylate - CTC chlorotetracycline - InsP₃ inositol 1,4,5-trisphosphate - RR ruthenium red - TMB-8 8-N-N-(diethylamino)-octyl-3,4,5-trimethoxybenzoate The author wishes to express his gratitude to the technical group of the Immunocytochemistry Unit for their help with the microinjection studies. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (grant A-7844).     

The polyphosphoinositides,phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate,are present in nuclei isolated from carrot protoplast

Summary Nuclei were isolated from carrot protoplasts and the distribution of [³H]inositol-labeled phospholipids was analyzed by thinlayer chromatography. Phosphatidylinositol (PI), lysophos-phatidylinositol (LPI), phosphatidylinositol monophosphate (PIP), lysophosphatidylinositol monophosphate (LPIP), and phosphatidylinositol bisphosphate (PIP₂) were 55.7%, 12.3%, 5.0%, 11.5%, and 3.6% of the respective [³H]inositol-labeled lipids recovered from the nuclear fraction. While both the plasma membrane and nuclear fraction contained polyphosphoinositides, the distribution of the phosphoinositides and the amount of inositol-labeled lipid were distinct. For example, the nuclear fraction had a higher percentage of LPI and PIP₂ and less PI and LPIP than the plasma membrane fraction. The amount of [³H]inositol-labeled lipid recovered from the nuclear fraction per mg protein was an order of magnitude lower than that recovered from either the plasma membrane of lower phase fraction isolated by aqueous two-phase partitioning, or from whole cells and protoplasts. In addition, when the ratio of the [³H]inositol-labeled lipid was compared to total [¹⁴C]myristate-labeled lipid recovered there was three to ten fold less [³H] relative to [¹⁴C] in the nuclear fraction.These data indicate that while the polyphosphoinositides are a relatively high percentage of the inositol lipid in the nuclear fraction, the inositol lipid was only a small portion of the total lipid in the nuclei. Despite this low concentration of inositol lipid, when [ ³²P]-ATP was added to the isolated nuclei,³²P-labeled PIP and PIP₂ were synthesized. Thus, the carrot nuclei contained PI and PIP kinase as well as the polyphosphoinositides.Abbreviations PI phosphatidylinositol - LPI lysophosphatidylinositol - PIP phosphatidylinositol monophosphate - LPIP lysophosphatidylinositol monophosphate - PIP₂ phosphatidylinositol bisphosphate - DAG diacylglycerol - IP₃ inositol 1,4,5-trisphosphate     

Concanavalin A binding to Chlamydomonas eugametos flagellar proteins and its effect on sexual reproduction

The relative amounts of Concanavalin A (Con A) bound by gamete and vegetative flagella of both mating types (mt ⁺ and mt ^-) of Chlamydomonas eugametos were determined using ¹²⁵I-Con A. Con A agglutinated all cell types by cross-linking their flagella in a random manner. No correlation was found between the extent of Con A-binding and Con A-mediated isoagglutination. Con A inhibited the sexual interaction between gametes at various levels. In mt ⁺ gametes it blocked sexual agglutination, whereas in mt ^- gametes it prevented papillar fusion. By SDS-gel electrophoresis nine Con A-binding components were found to be present in flagella. However, it was not possible to allocate a role in sexual agglutination to any of these components since they were present in all cell types, including vegetative cells which are not able to sexually agglutinate.Abbreviations Con A concanavalin A - SDS sodium dodecyl sulphate - TB Tris buffer - PBS phosphate buffered saline - HRP horse radish peroxidase - SEM scanning electron microscope - PAS periodic acid Schiff     

Mitochondrial genome transmission in Chlamydomonas diploids obtained by sexual crosses and artificial fusions: Role of the mating type and of a 1 kb intron

Summary The linear mitochondrial DNAs of the two infertile algal species Chlamydomonas smithii and C. reinhardtii are co-linear with the exception of a 1 kb intron ( intron) located in the cytochrome b gene of C. smithii. C. smithii also possesses an additional HpaI restriction site (H marker) located in the COXI gene, about 5 kb from the intron. In reciprocal crosses, C. smithii (H ⁺⁺) × C. reinhardtii (H ^––), the intron is transmitted to all diploid progeny, whereas the H marker is frequently transmitted either biparentally or paternally depending on whether the C. smithii parent is maternal (mt ⁺) or paternal (mt ^–). In diploids resulting from artificial fusion between vegetative cells, the absolute transmission of a is accompanied by the frequent transmission of the H ⁺ marker, irrespective of the mating type of the parental strains. Finally, in reciprocal crosses between C. smithii (H ⁺⁺) and recombinant H ^–+ clones, the transmission of the H marker is predominantly paternal or biparental. These results allow us to conclude that (1) the a intron behaves as a group I intron whose unidirectional conversion influences the transmission of the H marker; and (2) the mt ^– paternal mitochondrial genome is transmitted more often than the mt ⁺. The mating type has no effect in diploids obtained by artificial fusion.     

Mating receptor complex in the flagellar membrane of Chlamydomonas eugametos gametes

Summary The protein composition of the flagellar membrane of C. eugametos mt ^–gametes was analyzed using SDS-polyacrylamide gel electrophoresis. The association of the proteins with the membrane was assessed by differential extraction and an assay for glycosylation. Particular attention was paid to integral membrane proteins that could be associated with the mt ^– agglutinin, the membrane-bound sexual receptor by which the mt ^– gamete binds to its mt ⁺ partner. This agglutinin is a peripheral membrane glycoprotein and must be bound to the flagellar surface by an integral membrane anchor protein that connects the agglutinin with the cell's interior. Immunoaffinity chromatography was performed using Mab 66.3, a monoclonal antibody specific for the mt ^– agglutinin, in order to isolate protein complexes consisting of agglutinin molecules and associated components. Only one integral membrane glycoprotein (M_r = 125 kDa) was isolated that has an association with the agglutinin. It did not bind Mab 66.3, but did bind the lectin wheat germ agglutinin. This was an expected property of the membrane anchor protein because previous research (Kooijman et al. 1989) has shown that cross-linking a WGA-binding glycoprotein by this lectin induces sexual responses that are similar to those induced by agglutinin-agglutinin interactions during mating. We conclude that the 125-kDa glycoprotein is the membrane anchor for the agglutinin.Abbreviations BSA Bovine serum albumin - CBB Coomassie Brilliant Blue - CHAPS 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate - GTC guanidine thiocyanate - mt ^–/mt ⁺ mating type minus/plus - PAS periodic acid Schiff - PBS phosphate buffered saline - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TBS TRIS-buffered saline - WGA wheat germ agglutinin     

The effects of FMRFamide, 5-hydroxytryptamine and phorbol esters on the heart of the mussel Geukensia demissa

Summary The ventricle of the mussel Geukensia demissa is inhibited by 5-hydroxytryptamine and excited by the molluscan neuropeptide FMRFamide. Supra-threshold doses of amide result in marked positive chronotropy and inotropy within 5–15 s. 5-Hydroxytryptamine at 10^-8 M produces diastolic arrest within 10 s. A 1-min exposure to FMRFamide (5 · 10^-8 M) results in a small increase in the cytoplasmic levels of adenosine 3,5-cyclic monophosphate; shorter or longer exposures have no effect. The cAMP content of ventricles incubated in 5 · 10^-8 M 5-hydroxytryptamine for 1 min decreases by 2.3 pmol/mg protein; longer or shorter incubations have no effect. Treatment with forskolin results in 3-or 4-fold increases in adenosine 3,5-cyclic monophosphate, but forskolin has no effect on the mechanical activity of the ventricle. The levels of inositol monophosphate, inositol 1,4-diphosphate, and inositol 1,4,5-triphosphate in tissues exposed to 5-hydroxytryptamine are not different from levels in control tissues. FMRFamide decreases the levels of these phosphoinositides by 50% or more. Lower concentrations of phorbol 12,13-diacetate (10^-8 to 10^-7 M) and phorbol 12-myristate, 13-acetate (10^-6 M) cause positive chronotropy in the isolated ventricle; higher concentrations induce systolic arrest. These results suggest that the effects of 5HT on the ventricle are not mediated by adenosine 3,5-cyclic monophosphate or inositol 1,4,5-triphosphate. The effects of FMRFamide may involve a decrease in inositol 1,4,5-triphosphate. The effects of amide may involve a decrease in inositol 1,4,5-triphosphate. The response of the ventricles to phorbol esters suggest that protein kinase C may be involved in the regulation of cardiac contractility.Abbreviations cAMP adenosine 3,5-cyclic monophosphate - DMA dimethylformamide - DMSO dimethylsulfoxide - FMRFamide Phenylalanyl-methionyl-arginyl-phenylalanylamide - 5HT 5-hydroxytryptamine - IP inositol monophosphate - IP₂ inositol 1,4-diphosphate - IP₃ inositol 1,4,5-triphosphate - PDA phorbol 12,13-diacetate - PMA phorbol 12-myristate, 13-acetate - SW sea water Present address: MSU; E.M. Center, Memphis, TN 38152, USA     

An inositol 1,4,5-trisphosphate-6-kinase activity in pea roots

A soluble extract from pea (Pisum sativum L.) roots, when incubated with ATP and inositol 1,4,5-trisphosphate, produced an inositol tetrakisphosphate. The chromatographic properties of this inositol tetrakisphosphate, and of the products formed by its chemical degradation, identify it as inositol 1,4,5,6-tetrakisphosphate. No evidence was obtained for a 3-phosphorylation of inositol 1,4,5-trisphosphate. The importance of these observations with respect to inositol phosphates and calcium signalling in higher plants, is discussed.Abbreviations HPLC high-performance liquid chromatography - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - InsP₄ inositol tetrakisphosphate J.A.C. gratefully acknowledges support from the Agricultural and Food Research Council, U.K., Plant Molecular Biology Initiative.     

Evidence for a functional membrane barrier in the transition zone between the flagellum and cell body of Chlamydomonas eugametos gametes

Evidence is presented which supports the concept of a functional membrane barrier in the transition zone at the base of each flagellum of Chlamydomonas eugametos gametes. This makes it unlikely that agglutination factors present on the surface of the cell body can diffuse or be transported to the flagellar membrane. The evidence is as follows: 1) The glycoprotein composition of the flagellar membrane is very different to that of the cell-body plasma membrane. 2) The flagella of gametes treated with cycloheximide, tunicamycin or , -dipyridyl become non-agglutinable but the source of agglutination factors on the cell body is not affected. 3) Even under natural conditions when the flagella are non-agglutinable, for example in vis-à-vis pairs or in appropriate cell strains that are non-agglutinable in the dark, the cell bodies maintain the normal complement of active agglutinins. 4) When flagella of living cells are labeled with antibodies bound to fluorescein, the label does not diffuse onto the cell-body surface. 5) When gametes fuse to form vis-à-vis pairs, the original mating-type-specific antigenicity of each cell body is slowly lost (probably due to the antigens diffusing over both cell bodies), while the specific antigenicity of the flagellar surface is maintained. Even when the flagella of vis-à-vis pairs are regenerated from cell bodies with mixed antigenicity, the antigenicity of the flagella remains matingtype-specific. 6) Evidence is presented for the existence of a pool of agglutination factors within the cell bodies but not on the outer surface of the cells.Abbreviations and symbols CHI cycloheximide - GTC guaniline thiocyanate - mt ⁺/mt ^- mating type plus or minus - PAS Periodic-acid-Schiff reagent - SDS sodium dodecyl sulphate