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Despite its many advantages as an experimental system for the study of the epigenesis of polarity, it is obvious that the fucoid zygote also presents many problems. The development of polarity proceeds largely independently of direct gene action and thus may be considered a problem in cellular physiology. Ca2+ appears to play an important role in the process, but the optical properties of the zygotes (opacity and autofluorescence) hamper the use of modern methods of visualizing the distribution of Ca2+ and other ions. Likewise, other approaches, such as injection of fluorescent-labeled G-actin, in order to study the dynamics of actin filaments, are subject to the same limitations. It may be that the application of two-photon microscopy will enable experimenters to avoid some of these problems. This technique uses excitation wavelengths that are twice the wavelength of maximum absorption by fluorophores, and sufficient photon density for absorption is achieved only in a thin section. The fucoid zygotes are considerably more transparent to longer wavelengths, so attenuation of the exciting light and autofluorescence should be significantly reduced. Perhaps we will then be able to see further into these opaque cells. Another problem concerns the use of different species and genera. This may be unavoidable; for example, those of us who are land-locked tend to rely on Pelvetia, as it travels and stores better than the various species of Fucus and is less seasonal. Our colleagues fortunate enough to work near the ocean prefer to use the species that are locally available. Nevertheless, it is important to be careful about cross-genus and cross-species generalizations. While it is unlikely, based on what we know, that there are fundamental differences in physiological mechanisms among species, there may be small but still important differences in details. Obviously, investigators should directly compare results in more than one species whenever possible. The area of greatest disagreement, perhaps, concerns the mechanism of polarity formation, as opposed to its overt manifestation, germination. Are Ca2+ and actin involved or not? Assuming Ca2+ is involved, is the source internal or external? One basis for the different findings may be the differences in the strength of the polarizing signal provided to the zygotes. Clearly, the cells have powerful mechanisms for amplifying a faint asymmetry and developing an axis in response to an external signal. Furthermore, the fucoids generally develop in the intertidal zone and thus must be adapted to meeting the challenge of a widely varying external environment. They may have alternate mechanisms for responding to unilateral light. We have adopted the approach of presenting the cells with a fairly weak light signal--the minimum required to induce a considerable degree of organization of a population of zygotes. We then determine the effects of various inhibitors on photopolarization. One advantage of this approach is that it has allowed us to find treatments that increase the sensitivity of the zygotes to light, something that would not be possible if the untreated controls were fully polarized. Some of the differences between our results and those of others may be related to their use of a stronger light stimulus. It may be that if given a strong stimulus, a sufficient trace is left in the cells so that they can organize an axis when an inhibitor is removed. Careful consideration of this point may help to reconcile apparently contradictory findings. Despite these difficulties, the fucoid zygotes are likely to continue to be an important experimental system. Technology, including the development of more specific inhibitory reagents, may allow some of the shortcomings of the system to be overcome, and careful consideration of experimental conditions may resolve some of the points of disagreement.
Animal cells can change shape and move by using actin polymerization to drive plasma membrane protrusion. Giannone and coworkers (this issue of Cell) describe how cells periodically pull back on these protrusions in order to sense and respond to the rigidity of their environment.
The effect of experimental procedures designed to modify an intracellular phosphoinositide signalling pathway, which may be instrumental in the photophobic response of the protozoan ciliate Blepharisma japonicum, has been investigated. To assess this issue, the latency time of the photophobic response and the cell photoresponsiveness have been assayed employing newly developed computerized videorecording and standard macro-photographic methods. Cell incubation with neomycin, heparin and Li+, drugs known to greatly impede phosphoinositide turnover, causes evident dose-dependent changes in cell photomotile behaviour. The strongest effect on photoresponses is exerted by neomycin, a potent inhibitor of polyphosphoinositide hydrolysis. The presence of micromolar concentrations of neomycin in the cell medium causes both prolongation of response latency and decrease of cell photoresponsiveness. Neomycin at higher concentrations (> 10 microM) abolishes the cell response to light at the highest applied intensity. A slightly lower inhibition of cell responsiveness to light stimulation and prolongation of response latency are observed in cells incubated in the presence of heparin, an inositol trisphosphate receptor antagonist. Lithium ions, widely known to deplete the intracellular phosphoinositide pathway intermediate, inositol trisphosphate, added to the cell medium at millimolar level, also cause a slowly developing inhibitory effect on cell photoresponses. Mastoparan, a specific G-protein activator, efficiently mimics the effect of light stimulation. In dark-adapted ciliates, it elicits ciliary reversal with the response latency typical for ciliary reversal during the photophobic response. Sustained treatment of Blepharisma cells with mastoparan also suppresses the photoresponsiveness, as in the case of cell adaptation to light during prolonged illumination. The mastoparan-induced responses can be eliminated by pretreatment of the cells with neomycin. Moreover, using antibodies raised against bovine transducin, a cross-reacting protein with an apparent molecular mass of about 55 kDa in the Blepharisma cortex fraction is detected on immunoblots. The obtained results indirectly suggest that the changes in internal inositol trisphosphate level, possibly elicited by G-protein-coupled phospholipase C, might play a role in the photophobic response of Blepharisma. However, further experiments are necessary to clarify the possible coupling between the G-protein and the putative phospholipase C.
Phototransduction in vertebrate photoreceptor cells mediated by rhodopsin is one of the most comprehensively examined G protein-coupled receptor (GPCR) signaling pathways. The signal transduction pathway can be mapped from the initial absorption of light to conformational changes within rhodopsin, through activation of the G protein transducin, and to the ultimate closure of the cation cGMP-gated channels in the plasma membrane. Furthermore, phototransduction has become an intensely studied model system for understanding the desensitizing processes that allow reduced non-linear responses of photoreceptor cells to increasing levels of illumination. Although some general themes appear to occur in GPCR systems, the details of these desensitizing processes are likely to be specific to each of the receptors. These differences are attributed to the fact that each receptor has unique kinetic constraints, amplification levels, tolerance to basal constitutive activity, intracellular internalization and recycling, redundancy of isoforms, and morphologies of the cell of their expression. One of the biochemical processes that are believed to be a common part of this desensitization of the GPCR-mediated cascade is receptor phosphorylation catalyzed by members of a small family of the GPCR kinases. The enzymatic, physiological and genetic aspects of rhodopsin phosphorylation and rhodopsin kinase have been characterized extensively over the last 30 yr. However, new structurally based approaches to examining rhodopsin kinase and rhodopsin phosphorylation are still awaiting further investigations. We present here a summary of the current understanding of rhodopsin phosphorylation and the properties of rhodopsin kinase, along with some expectations of future investigations into these topics.
Plants have evolved sophisticated sensing mechanisms that operate through phytochromes, perceiving changes in the red:far-red ratio, which trigger morphological changes to avoid shade. The shade-avoidance response essentially redirects resources and growth potential from the leaf and storage organs into increased extension growth to optimize light capture by plants. Recent studies implicate ATHB-2, a homeodomain-leucine zipper transcription factor, as a regulator of shade-avoidance responses and establish a strong link between this factor and auxin signaling. The action of ATHB-2 is likely to generate changes in auxin distribution that produce distinct but coordinated effects on different cell types across the plant. Future studies should highlight how polarity of auxin transport is altered in response to light-quality changes.
The intrinsic tryptophan fluorescence of the alpha subunit of transducin (alpha T) has been shown to be sensitive to the binding of guanine nucleotides, with the fluorescence being enhanced by as much as 2-fold upon the binding of GTP or nonhydrolyzable GTP analogues [cf. Phillips and Cerione (1988) J. Biol. Chem. 263, 15498-15505]. In this work, we have used these fluorescence changes to analyze the kinetics for the activation (GTP binding)-deactivation (GTPase) cycle of transducin in a well-defined reconstituted phospholipid vesicle system containing purified rhodopsin and the alpha T and beta gamma T subunits of the retinal GTP-binding protein. Both the rate and the extent of the GTP-induced fluorescence enhancement are dependent on [rhodopsin], while only the rate (and not the extent) of the GTP gamma S-induced enhancement is dependent on the levels of rhodopsin. Comparisons of the fluorescence enhancements elicited by GTP gamma S and GTP indicate that the GTP gamma S-induced enhancements directly reflect the GTP gamma S-binding event while the GTP-induced enhancements represent a composite of the GTP-binding and GTP hydrolysis events. At high [rhodopsin], the rates for GTP binding and GTPase are sufficiently different such that the GTP-induced enhancement essentially reflects GTP binding. A fluorescence decay, which always follows the GTP-induced enhancement, directly reflects the GTP hydrolytic event. The rate of the fluorescence decay matches the rate of [32P]Pi production due to [gamma-32P]GTP hydrolysis, and the decay is immediately reversed by rechallenging with GTP. The GTP-induced fluorescence changes (i.e., the enhancement and ensuing decay) could be fit to a simple model describing the activation-deactivation cycle of transducin. The results of this modeling suggest the following points: (1) the dependency of the activation-deactivation cycle on [rhodopsin] can be described by a simple dose response profile; (2) the rate of the rhodopsin-stimulated activation of multiple alpha T(GDP) molecules is dependent on [rhodopsin] and when [alpha T] greater than [rhodopsin], the activation of the total alpha T pool may be limited by the rate of dissociation of rhodopsin from the activated alpha T(GTP) species; and (3) under conditions of optimal rhodopsin-alpha T coupling (i.e., high [rhodopsin]), the cycle is limited by GTP hydrolysis with the rate of Pi release, or any ensuing conformational change, being at least as fast as the hydrolytic event.
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Photoactive yellow protein, a small, water-soluble blue-light absorbing photoreceptor protein from Ectothiorhodospira(Halorhodospira)[space]halophila has a structure with two hydrophobic cores, of which the main one houses its light-sensitive chromophore (p-coumaric acid), separated by a central [small beta]-sheet. This photoreceptor protein contains a single tryptophan residue (W119) that is situated at the interface between the central beta-sheet and its N-terminal cap. The fluorescence properties of W119 in the dark state pG (lambda(max)= 328 nm; Phi(fl)= 0.01; nearly pH-independent) are typical for a buried tryptophan in a hydrophobic environment with significant quenching by nearby amino acid residues. Signalling state formation leads to pH-dependent fluorescence changes: At pH values <6.5 the fluorescence emission increases, with a minor blue shift of the emission maximum. Above this pH, the emission maximum of the tryptophan shifts considerably to the red, whereas its total intensity decreases. These results further support the contention that signalling state formation in PYP leads to significant changes in the structure of this protein, even at sites that are at a considerable distance from the chromophore. The nature of these changes in pB, however, depend upon the pH imposed upon the protein: At slightly alkaline pH, which presumably is closest to the pH to which this protein is exposed in vivo, these changes lead to an exposure of the part of the central beta-sheet harbouring W119. At slightly acidic pH the polarity of the environment of W119 is hardly affected by the formation of the signalling state but the quenching of its fluorescence emission, possibly by nearby amino acids, is reduced. On the other hand, its accessibility for quenching by small molecules in the solution is enhanced at acidic and alkaline pH in the signalling state (pB) compared to the dark state (pG). This latter observation points towards a more flexible structure of the N-terminal cap, having a looser interaction with the central beta-sheet in pB.
In view of the ability of calmodulin to bind vasoactive intestinal peptide (VIP) and growth-hormone-releasing factor (GRF) with high affinity [Stallwood, Brugger, Baggenstoss, Stemmer, Shiraga, Landers and Paul (1992) J. Biol. Chem. 267, 19617-19621], the effects of these neuropeptides on a model calmodulin-dependent enzyme, myosin light-chain kinase (MLCK), were studied. Both peptides were potent inhibitors of MLCK activity. The inhibition of enzyme activity by VIP and GRF was progressively overcome with increasing calmodulin concentrations, with no inhibition observed at a saturating calmodulin concentration. Nanomolar concentrations of MLCK blocked the formation of calmodulin-[125I-Tyr10]VIP complexes. These data provide support for a functional role of VIP and GRF binding by calmodulin.
Plant tetrapyrroles are the most abundant biomolecules on the earth and are cofactors of many apoproteins essential for plant function. The four end-products sirohaem, chlorophyll, haem and phytochromobilin are synthesized by a common branched pathway, which is tightly regulated to ensure a continuous supply to the cognate apoproteins. This may induce strong competition between different branches of the pathway for common substrates. In addition, the intermediates, which are phototoxic, must not be allowed to accumulate in the cell. The major control points are during the synthesis of the initial precursor, ALA (5-aminolaevulinic acid), and at the branch points for the insertion of metal ions into the porphyrin macrocycle. A recent study has also suggested that tetrapyrroles are involved in the communication between the chloroplast and the nucleus, strengthening the necessity for tight regulation. However, intermediates remain difficult to quantify mainly due to their low content and the different properties of the intermediates. In this paper, we summarize the regulation of this pathway and we detail why it is important to have an accurate method for the determination of tetrapyrroles in plants.
Illumination of vertebrate rod photoreceptors leads to a decrease in the cytoplasmic cGMP concentration and closure of cyclic nucleotide-gated (CNG) channels. Except for Ca(2+), which plays a negative feedback role in adaptation, and 11-cis-retinal, supplied by the retinal pigment epithelium, all of the biochemical machinery of phototransduction is thought to be contained within rod outer segments without involvement of extrinsic regulatory molecules. Here we show that insulin-like growth factor-I (IGF-I), a paracrine factor released from the retinal pigment epithelium, alters phototransduction by rapidly increasing the cGMP sensitivity of CNG channels. The IGF-I-signaling pathway ultimately involves a protein tyrosine phosphatase that catalyzes dephosphorylation of a specific residue in the alpha-subunit of the rod CNG channel protein. IGF-I conjointly accelerates the kinetics and increases the amplitude of the light response, distinct from events that accompany adaptation. These effects of IGF-I could result from the enhancement of the cGMP sensitivity of CNG channels. Hence, in addition to long-term control of development and survival of rods, growth factors regulate phototransduction in the short term by modulating CNG channels.
Honey bee photoreceptors contain large sacs of endoplasmic reticulum (ER) that can be located unequivocally in freeze-dried cryosections. The elemental composition of the ER was determined by electron probe x-ray microanalysis and was visualized in high-resolution x-ray maps. In the ER of dark-adapted photoreceptors, the Ca concentration was 47.5 +/- 1.1 mmol/kg (dry weight) (mean +/- SEM). During a 3-sec nonsaturating light stimulus, approximately 50% of the Ca content was released from the ER. Light stimulation also caused a highly significant increase in the Mg content of the ER; the ratio of Mg uptake to Ca released was approximately 0.7. Our results show unambiguously that the ER is the source of Ca2+ release during cell stimulation and suggest that Mg2+ can nearly balance the charge movement of Ca2+.
Insights into structure-function relations of many proteins opens the possibility of engineering peptides to selectively interfere with a protein's activity. To facilitate the use of peptides as probes of cellular processes, we have developed caged peptides whose influence on specific proteins can be suddenly and uniformly changed by near-UV light. Two peptides are described which, on photolysis of a caging moiety, block the action of calcium-calmodulin or myosin light chain kinase (MLCK). The efficacy of theses peptides is demonstrated in vitro and in vivo by determining their effect before and after photolysis on activities of isolated enzymes and cellular functions known to depend on calcium-calmodulin and MLCK. These caged peptides each were injected into motile, polarized eosinophils, and when exposed to light promptly blocked cell locomotion in a similar manner. The results indicate that the action of calcium-calmodulin and MLCK, and by inference myosin II, are required for the ameboid locomotion of these cells. This methodology provides a powerful means for assessing the role of these and other proteins in a wide range of spatio-temporally complex functions in intact living cells.
Light-dependent activation of thylakoid protein phosphorylation regulates the energy distribution between photosystems I and II of oxygen-evolving photosynthetic eukaryotes as well as the turnover of photosystem II proteins. So far the only known effect of light on the phosphorylation process is the redox-dependent regulation of the membrane-bound protein kinase(s) activity via plastoquinol bound to the cytochrome bf complex and the redox state of thylakoid dithiols. By using a partially purified thylakoid protein kinase and isolated native chlorophyll (chl) a/b light-harvesting complex II (LHCII), as well as recombinant LHCII, we find that illumination of the chl-protein substrate exposes the phosphorylation site to the kinase. Light does not activate the phosphorylation of the LHCII apoprotein nor the recombinant pigment-reconstituted complex lacking the N-terminal domain that contains the phosphothreonine site. The suggested light-induced conformational change exposing the N-terminal domain of LHCII to the kinase is evidenced also by an increase in its accessibility to tryptic cleavage after light exposure. Light activates preferentially the trimeric form of LHCII, and the process is paralleled by chl fluorescence quenching. Both phenomena are slowly reversible in darkness. Light-induced exposure of the LHCII N-terminal domain to the endogenous protein kinase(s) and tryptic cleavage occurs also in thylakoid membranes. These results demonstrate that light may regulate thylakoid protein phosphorylation not only via the signal transduction chain connecting redox reactions to the protein kinase activation, but also by affecting the conformation of the chl-protein substrate.
The diffusible platelet stimuli ADP and thromboxane A(2) activate multiple G protein-mediated signaling pathways and function as important secondary mediators of platelet activation as they are released from activated platelets. Because they can also increase their own formation and release, their effects are amplified; eventually, all major G protein-mediated signaling pathways are activated. The multiple positive feedback mechanisms operating during platelet activation have obscured the exact analysis of the roles individual G protein-mediated signaling pathways play during the platelet activation process. In this report, we show that platelets lacking G(q) and G(13) are completely unresponsive to diffusible stimuli such as ADP, thromboxane A(2), or thrombin, even when applied at very high concentrations in combination, whereas all stimuli are able to induce platelet aggregation, shape change, and RhoA activation in platelets lacking only one Galpha subunit. This shows that G(q) or G(13) is required to induce some platelet activation, whereas the activation of G(i)-mediated signaling alone is not sufficient to induceactivation of mouse platelets. In addition, platelets lacking Galpha(q) and Galpha(13) adhered normally to collagen under high shearbut did not aggregate any more in response to collagen, indicating that collagen-induced platelet activation but not platelet adhesion requires intact G protein-mediated signaling pathways.
The influence of daily photoperiod (8, 16, 24 h) on eight clones of Spirodela polyrhiza was tested in two different nutrient media. The number of vegetative fronds and resting turions formed after 50 days of cultivation were scored. The specific turion yield (STY; number of turions formed per vegetative frond) was used to evaluate the effectiveness of turion formation of the tested clones. All clones formed turions in both nutrient media. The STY varied substantially between the different clones, ranging from 0.22 +/- 0.03 (clone SC from Cuba) to 3.9 +/- 0.3 (clone 9256 from Finland) in continuous light. The STY increased with increasing duration of the photoperiod. This increase may have been due to the extended period of photosynthesis rather than that of a photoperiodic long-day response. Shorter photoperiods did not stimulate turion formation in any of the clones. S. polyrhiza is a day-neutral plant with respect to turion formation, as noted previously (Appenroth et al. 1990. Annals of Botany 66: 163-168). In accordance with this conclusion, no correlation was detected between the STY and the latitude at which the clones occur naturally. Environmental factors other than shortening of photoperiods seem to be effective in signalling seasonal changes of growth conditions in advance to S. polyrhiza.
An unknown signal at 2.93 ppm in 1H-NMR spectra of rice, Oryza sativa, was assigned to the methyl groups of sulphur-methylmethionine (SMM), thereby devising a new method for the determination of this compound. Rice seedlings growing aerobically in the dark and in the light engaged for the synthesis of SMM an amount of Met corresponding to 23 and 8%, respectively, of the total seed reserves of this amino acid. In etiolated shoots, SMM reached 1.2 micromol g(-1) fresh weight, an unusually high level in vegetative tissues of wild-type plants. This is compared to a value of 0.4 micromol g(-1) fresh weight in green tissues. A decreased demand for Met during growth caused the higher accumulation of SMM in etiolated, rather than green, tissues. At the same time, dark seedlings were endowed with a readily utilizable and translocable alternative form of Met, as shown by retrieval of SMM from the coleoptile. The importance of methyl group storage in SMM is shown by comparison with choline and choline phosphate pools.