Identification of the agg1 mutation responsible for negative phototaxis in a “wild-type” strain of Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a model organism for various studies in biology. CC-124 is a laboratory strain widely used as a wild type. However, this strain is known to carry agg1 mutation, which causes cells to swim away from the light source (negative phototaxis), in contrast to the cells of other wild-type strains, which swim toward the light source (positive phototaxis). Here we identified the causative gene of agg1 (AGG1) using AFLP-based gene mapping and whole genome next-generation sequencing. This gene encodes a 36-kDa protein containing a Fibronectin type III domain and a CHORD-Sgt1 (CS) domain. The gene product is localized to the cell body and not to flagella or basal body.     

Hydrolysis of cyclic GMP in rat peritoneal macrophages

Intact rat peritoneal macrophages (rPM) treated with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases (PDEs), accumulated more cGMP than untreated cells. A PDE activity toward [(3)H]cGMP was detected in the soluble and particulate fractions of rPM. The hydrolysis of cGMP was Ca(2+)/calmodulin-independent but increased in the presence of cGMP excess. Similar results were obtained when [(3)H]cAMP was used as a substrate. The hydrolytic activity towards both nucleotides was inhibited in the presence of IBMX. Therefore, the PDEs of families 2, 5, 10 and 11 are potential candidates for cGMP hydrolysis in the rPM. They may not only regulate the cGMP level in a feedback-controlled way but also link cGMP-dependent pathways with those regulated by cAMP.     

Activation of Chlamydomonas rhodopsin in vivo does not require isomerization of retinal

The unicellular eukaryote Chlamydomonas reinhardtii is a phototactic alga that swims toward or away from light, using rhodopsin as the photopigment. The activity of retinal analogues was tested in the mutant FN68, which has high phototactic sensitivity only after incubation with retinal or analogues of retinal. Analogues prevented from isomerizing about the 7-ene, 9-ene, 11-ene, 13-ene, or 15-ene (C = N+H) bonds retained full activity. Also, bleaching, protonation of the N, and a stable geometrically altered chromophore are not required for full activity. An attractive hypothesis is that charge redistribution in the excited state of retinal directly triggers the activity of rhodopsin.     

Two flagellar genes, AGG2 and AGG3, mediate orientation to light in Chlamydomonas

Ciliary membranes have a large repertoire of receptors and ion channels that act to transduce information from the environment to the cell. Chlamydomonas offers a tractable system for dissecting the transport and function of ciliary and flagellar membrane proteins. Isolation of ergosterol and sphingolipid-enriched Chlamydomonas flagellar membrane domains identified potential signaling molecules by mass spectroscopy. These include a membrane protein and a matrix flavodoxin protein that are encoded by the AGG2 and AGG3 genes, respectively. Agg2p localizes to the proximal flagellar membrane near the basal bodies. Agg3p is distributed throughout the flagellar matrix, with an increased concentration in the proximal regions where Agg2p is located. Chlamydomonas cells sense light by using a microbial-type rhodopsin , transduce a signal from the cell body to the flagella, and alter the waveform of the flagella to turn a cell toward the light. Protein depletion by RNA interference reveals that both AGG gene products play roles in the orientation of cells to a directional light source. The depleted strains mimic the phenotype of the previously identified agg1 mutant, which swims away from light. We propose that the localization of Agg2p and Agg3p to the proximal region of the flagella may be important for interpreting light signals.     

Altering cAMP levels within a central pattern generator modifies or disrupts rhythmic motor output

Cyclic AMP is a second messenger that has been implicated in the neuromodulation of rhythmically active motor patterns. Here, we tested whether manipulating cAMP affects swim motor pattern generation in the mollusc, Tritonia diomedea. Inhibiting adenylyl cyclase (AC) with 9-cyclopentyladenine (9-CPA) slowed or stopped the swim motor pattern. Inhibiting phosphodiesterase with 3-isobutyl-1-methylxanthine (IBMX) or applying dibutyryl-cAMP (dB-cAMP) disrupted the swim motor pattern, as did iontophoresing cAMP into the central pattern generator neuron C2. Additionally, during wash-in, IBMX sometimes temporarily produced extended or spontaneous swim motor patterns. Photolysis of caged cAMP in C2 after initiation of the swim motor pattern inhibited subsequent bursting. These results suggest that cAMP levels can dynamically modulate swim motor pattern generation, possibly shaping the output of the central pattern generator on a cycle-by-cycle basis.     

Effects of UV-B on motility and photobehavior in the green flagellate,Euglena gracilis

UV-B inhibits the motility of the green flagellate, Euglena gracilis, at fluences rates higher than those expected to occur in the natural sunlight even when the stratospheric ozone layer is partially reduced by manmade pollutants. The phototactic orientation of the cells, however, is drastically impaired by only slightly enhanced levels of UV-B irradiation. Since only negative phototaxis (movement away from a strong light source) is impaired while positive phototaxis (movement toward a weak light source) is not, the delicate balance by which the organisms adjust their position in their habitat is disturbed. Under these conditions the cells are unable to retreat from hazardous levels of radiation and are eventually killed not by the UV-B irradiation but by photobleaching of their photosynthetic pigments in the strong daylight at the surface.     

Bradykinin inhibition of cyclic AMP accumulation in D384 astrocytoma cells. Evidence against a role of cyclic GMP.

The present studies were performed in order to examine the possible role of cyclic GMP-stimulated phosphodiesterase (cGMP-PDE) activity in the inhibitory action of the inflammatory peptide bradykinin on cyclic AMP (cAMP) accumulation in D384 cells. Bradykinin decreased the forskolin-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor rolipram, and caused a transient 50% rise in cellular cGMP in the presence of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Both basal and bradykinin-stimulated cGMP accumulation were about 8 times higher in the presence of IBMX than in the presence of rolipram. Sodium nitroprusside, which caused a 20-70-fold increase in cGMP levels reduced forskolin stimulated cAMP accumulation, whereas hydroxylamine, which maximally caused a 16-fold increase in cGMP, did not. 8-bromo-cGMP or dibutyryl cGMP had no effect on cAMP accumulation induced by forskolin. The inhibitory effect of nitroprusside was totally reversed by blocking the soluble guanylate cyclase activity by methylene blue treatment; however, the inhibitory action of bradykinin on cAMP accumulation was not changed by this treatment. Additionally, inhibition of nitric oxide synthesis, which is known to be regulated by Ca2+ and in turn stimulates cGMP production, by N omega-nitro-L-arginine (L-NAME) treatment did not alter the inhibitory effect of bradykinin on forskolin-induced cAMP accumulation. These results indicate that large increases in cGMP may regulate cAMP via cGMP-PDE whereas the small increase induced by bradykinin is insufficient and that cGMP is not involved in the inhibitory action of bradykinin on cAMP levels in D384 cells.     

Photoactivated adenylyl cyclase controls phototaxis in the flagellate Euglena gracilis

Euglena gracilis, a unicellular freshwater protist exhibits different photomovement responses, such as phototaxis (oriented movement toward or away from the light source) and photophobic (abrupt turn in response to a rapid increase [step-up] or decrease [step-down] in the light fluence rate) responses. Photoactivated adenylyl cyclase (PAC) has been isolated from whole-cell preparations and identified by RNA interference (RNAi) to be the photoreceptor for step-up photophobic responses but not for step-down photophobic responses (M. Iseki, S. Matsunaga, A. Murakami, K. Ohno, K. Shiga, C. Yoshida, M. Sugai, T. Takahashi, T. Hori, M. Watanabe [2002] Nature 415: 1047-1051). The present study shows that knockdown of PAC by RNAi also effectively suppresses both positive and negative phototaxis, indicating for the first time that PAC or a PAC homolog is also the photoreceptor for photoorientation of wild-type E. gracilis. Recovery from RNAi occurred earlier for step-up photophobic responses than for positive and negative phototaxis. In addition, we investigated several phototaxis mutant strains of E. gracilis with different cytological features regarding the stigma and paraxonemal body (PAB; believed to be the location for the phototaxis photoreceptor) as well as Astasia longa, a close relative of E. gracilis. All of the E. gracilis mutant strains had PAC mRNAs, whereas in A. longa, a different but similar mRNA was found and designated AlPAC. Consistently, all of these strains showed no phototaxis but performed step-up photophobic responses, which were suppressed by RNAi of the PAC mRNA. The fact that some of these strains possess a cytologically altered or no PAB demonstrates that at least in these strains, the PAC photoreceptor responsible for the step-up photophobic responses is not located in the PAB.     

Exogenous cAMP and cGMP modulate branching in fusarium graminearum.

A study was made of the effects of adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP) and choline on the morphology and growth of a wild-type strain (A 3/5) and a highly branched, 'colonial' mutant strain (C106) of Fusarium graminearum. Addition of up to 50 mM-cAMP or cGMP to the medium had no effect on the specific growth rate of strain A 3/5. For strain A 3/5, but not for strain C106, exogenous cAMP caused significant decreases in both mean hyphal extension rate (E) and hyphal growth unit length (G), i.e. cAMP caused mycelia of strain A 3/5 to branch profusely. By contrast, for both strains, cGMP caused significant increases in both E and G, i.e. exogenous cGMP caused mycelia to branch more sparsely. The effects of exogenous cGMP and choline in increasing E and G were synergistic, but the effects of cGMP and choline counteracted the effect of cAMP. The mutant phenotype of strain C106 was not correlated with altered levels of endogenous cAMP or cGMP.     

Role of cAMP in modulating intrafollicular progesterone levels and oocyte maturation in amphibians (Rana pipiens)

The role of cAMP in regulating follicular progesterone levels and oocyte maturation was investigated following in vitro culture of amphibian (Rana pipiens) ovarian follicles. Intrafollicular levels of cAMP were manipulated with the use of a stimulator of cAMP synthesis (forskolin) or by exogenous addition of cAMP alone or either of these in combination with an inhibitor of cAMP catabolism (3-isobutyl-1-methyl xanthine, IBMX). Follicular progesterone content was determined by RIA and oocyte maturation was assessed cytologically. In the presence of increasing doses of forskolin (0-3 microM), cAMP (0-3 mM), or dibutyryl cAMP (dbcAMP, 0-2.5 mM) increasing but low levels of progesterone were detected. Increasing doses of IBMX (0-0.09 mM) alone had no significant effect on follicular steroid content. Exogenous cAMP, dbcAMP, or IBMX (0.09 mM) suppressed hormone-induced oocyte maturation. Simultaneous exposure of follicles to increasing doses of both forskolin (0-3 microM) and IBMX (0-0.09 mM) markedly increased intrafollicular progesterone levels to those produced by frog pituitary homogenate (FPH). A marked increase in progesterone levels also occurred when follicles were exposed to exogenous cAMP (3 mM) and IBMX (0.09 mM). These results indicate that exogenous cAMP is incorporated by follicle cells and that forskolin effects are mediated through cAMP. Changes in follicular progesterone levels (increase and decrease) over time following FPH or cAMP manipulation (cAMP + IBMX or forskolin + IBMX) were essentially identical. In contrast to cAMP, cGMP was inactive in inhibiting hormone induced GVBD or stimulating follicular progesterone accumulation. Elevation of follicular and medium levels of progesterone resulting from FPH or cAMP stimulation required the presence of the somatic follicular cells. The decrease in follicular progesterone levels with prolonged culture was not associated with a corresponding increase in progesterone levels in the medium. The decrease in follicular progesterone levels appears to reflect steroid catabolism rather than loss of steroid to the culture medium. The results suggest that the level of intracellular cAMP in the follicle cells is modulated by the relative activity of the adenylate cyclase system and phosphodiesterase and that FPH can affect both components. Thus, intracellular levels of cAMP play a key role in regulating follicular progesterone levels and FPH action on the follicle cells. The steroidogenic capacity of follicle cells can be manipulated independently of FPH stimulation.     

Diverse directional changes of cGMP relative to cAMP in E. coli.

The relationships of the changes of cAMP and cGMP concentrations in $\text{E. coli}$ varied as a function of experimental conditions. (1) Cells starved for carbon source for a short time period had high cAMP and low cGMP concentrations. Addition of carbon source (succinate, glucose or α-methyl glucoside) led to a decrease in cAMP and an increase in cGMP (bi-directional change). (2) Washed cells starved for glucose for long time periods had low cAMP levels which did not change on glucose addition. Addition of succinate or glucose to such cells led to a transient increase in cGMP levels (uncoupled change). The cGMP concentration peaked at 15 minutes or 1 hour after glucose or succinate addition, respectively. (3) Sham shift-up experiments (addition of α-methyl glucoside to cultures growing in succinate) in $\text{E. coli}$ 1100 and CA 8000 showed decreases in cGMP levels in both strains; however, cAMP levels increased in the former (bi-directional change) and decreased in the latter (unidirectional change).     

The sll0886 gene, controlling light-activated heterotrophic growth, is involved in regulating phototaxis in cyanobacterium Synechocystis sp. PCC 6893

The sll0886 gene, controlling light-activated heterotrophic growth (LAHG), was tested for the role in regulating phototaxis in cyanobacterium Synechocystis sp. PCC 6803. Insertional inactivation of the gene in the genome of a wildtype strain did not affect positive (toward light) or negative (away from high light) phototaxis. However, cells lost motility when sll0886 inactivation was combined with the prqRL17Q mutation, which determined negative phototaxis at low light. Immotile cells with the prqRL17Q mutation and the inactivated sll0886 gene did not display any defect in the formation of type IV pili, essential for phototaxis. Hence, the function, rather than biogenesis, of pili was affected. It was concluded that the sll0886 gene, coding for a TPR family protein, is involved in controlling negative phototaxis of cyanobacteria at the level of photoreception and signal transduction and that its role is mediated by the unidentified redundant gene whose function is suppressed by the prqRL17Q mutation.     

The involvement of a protein kinase in phototaxis and gravitaxis of <Emphasis Type="Italic">Euglena gracilis</Emphasis>

The unicellular flagellate Euglena gracilis shows positive phototaxis at low-light intensities (<10 W/m²) and a negative one at higher irradiances (>10 W/m²). Phototaxis is based on blue light-activated adenylyl cyclases, which produce cAMP upon irradiation. In the absence of light the cells swim upward in the water column (negative gravitaxis). The results of sounding rocket campaigns and of a large number of ground experiments led to the following model of signal perception and transduction in gravitaxis of E. gracilis: The body of the cell is heavier than the surrounding medium, sediments and thereby exerts a force onto the lower membrane. Upon deviation from a vertical swimming path mechano-sensitive ion channels are activated. Calcium is gated inwards which leads to an increase in the intracellular calcium concentration and causes a change of the membrane potential. After influx, calcium activates one of several calmodulins found in Euglena, which in turn activates an adenylyl cyclase (different from the one involved in phototaxis) to produce cAMP from ATP. One further element in the sensory transduction chain of both phototaxis and gravitaxis is a specific protein kinase A. We found five different protein kinases A in E. gracilis. The blockage of only one of these (PK.4, accession No. EU935859) by means of RNAi inhibited both phototaxis and gravitaxis, while inhibition of the other four affected neither phototaxis nor gravitaxis. It is assumed that cAMP directly activates this protein kinase A which may in turn phosphorylate a protein involved in the flagellar beating mechanism.     

Phototaxis inEuglena gracilis: Effect of sodium azide and triphenylmethyl phosphonium ion on the photosensory transduction chain

Phototaxis in the flagellateEuglena gracilis was studied by means of a microvideographic analysis, and the light-induced directional movement was determined by computer-based statistical treatments. Lateral white light with an illuminance of 25 lx (0.105 Wm^–2) caused the cells to preferentially swim toward the light source (positive phototaxis), while an illuminance of 1,000 lx (4.2 Wm^–2) induced negative phototaxis. The lipophilic membranepenetrating cation triphenylmethyl phosphonium ion (TPMP⁺) specifically inhibited positive phototaxis, while it hardly affected negative phototaxis. The uncoupler sodium azide, on the other hand, impaired negative phototaxis substantially.     

Multidirectional phototaxis by Dictyostelium discoideum amoebae

Abstract Phototaxis by solitary Dictyostelium discoideum amoebae is known to be complex, the amoebae turning either towards or away from the light, depending on conditions such as light intensity. Having previously shown that amoebal phototaxis can be bidirectional (2 preferred directions either side of the light source), we now report the discovery of multidirectional phototaxis by D. discoideum amoebae, with up to 12 different preferred directions. As in the bidirectional case, multidirectional phototaxis depends on direction-dependent transitions from turning away from to turning towards the light source.     

Guinea-pig lung adenylyl and guanylyl cyclase and PDE activities associated with airway hyper- and hypo-reactivity following LPS inhalation

The relationships between changes in in vivo airway reactivity and levels cyclicAMP and cyclicGMP were determined in guinea-pig lungs after exposure to inhaled lipopolysaccharide (LPS). After LPS (30 microg.ml(-1), 1 h), guinea-pigs displayed in vivo airway hyperreactivity (AHR) at 1 h and hyporeactivity (AHOR) at 48 h, to inhaled (20 s) histamine (1 or 3 mM, respectively). Isoprenaline-stimulated cAMP or SNAP-stimulated cGMP were determined in the lungs isolated from guinea-pigs exposed to LPS inhalation to determine whether there was a relationship between AHR or AHOR and adenylyl/guanylyl cyclase and phosphodiesterase (PDE) activities. Assays were performed in the absence and presence of the non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Levels of cAMP and cGMP in its presence indicated adenylyl and guanylyl cyclase activities, respectively. The difference between cAMP and cGMP levels, in the absence and presence of IBMX, reflected relevant PDE activity. In vivo AHR was associated with increased PDE activity towards cAMP and cGMP (67 and 278%, respectively) and also increased adenylyl (47%) and guanylyl (210%) cyclase activities. In vivo AHOR at 48 h after LPS inhalation was also associated with raised cyclase activity (p < 0.05), whereas relevant PDE activity declined by 79 and 68%, compared with 48 h after vehicle. Although net stimulated cGMP levels increased during AHR and AHOR and net stimulated cAMP increased during AHOR, our index of PDE activity increased during AHR and decreased during AHOR. These results therefore support the rationale for the use of PDE-inhibitors in the treatment of respiratory diseases associated with AHR.     

Complex effects of inhibitors on cyclic GMP-stimulated cyclic nucleotide phosphodiesterase

We have investigated the effects of several phosphodiesterase inhibitors on the activity of a cGMP-stimulated cyclic nucleotide phosphodiesterase purified from calf liver supernatant. Theophylline, RO 20-1724, and MY 5445 were not effective inhibitors. With 0.5 microM [3H]cGMP as substrate or with 0.5 microM [3H]cAMP in the presence of 1 microM cGMP, activity was inhibited by papaverine, dipyridamole, isobutylmethylxanthine (IBMX), and cilostamide. With 0.5 microM [3H]cAMP as substrate, however, only cilostamide was inhibitory; papaverine, dipyridamole, and IBMX increased activity. The increase was dependent on both drug and substrate concentration with maximal stimulation (150-180%) at concentrations of cAMP between 0.5 and 2.5 microM. At higher cAMP concentrations, the three drugs were inhibitory; inhibition was maximal at approximately 40 microM and decreased at higher cAMP concentrations. Inhibition of cGMP hydrolysis was maximal at approximately 3 microM and decreased at higher concentrations. Papaverine, IBMX, dipyridamole, and cilostamide inhibited [3H] cGMP hydrolysis competitively with Ki values of 3, 6.5, 7, and 11.5 microM, respectively. Papaverine, IBMX, or dipyridamole reduced the Hill coefficient for cAMP hydrolysis from 1.8 to 1.1-1.2, and Lineweaver-Burk plots were linear or nearly linear. With cilostamide, however, Lineweaver-Burk plots remained curvilinear. Thus, three competitive inhibitors, papaverine, dipyridamole, and IBMX, can mimic substrate and effect allosteric transitions that increase catalytic activity, whereas another, cilostamide, apparently cannot. Differences in the actions of these inhibitors presumably reflect differences in the molecular requirements for effective interaction at catalytic and allosteric sites on phosphodiesterase, i.e. differences in the structure of these sites.