Autonomous regulation of free Ca2+ concentrations in isolated plant cell nuclei: a mathematical analysis

Experiments performed on nuclei isolated from animal or plant cells have provided evidence that the nucleus generates directly specific nucleoplasmic calcium transients in response to external stimuli. Recent data suggest that isolated plant nuclei might be considered as a closed system where the nuclear concentration of free calcium would be regulated by reversible movements between the nucleoplasm and nuclear stores. We have addressed the relevance of this hypothesis by developing a mathematical approach to simulate nucleoplasmic calcium dynamics generated under various pH and temperature conditions. Here, we show that the experimental results could be explained provided that calcium channels as well as systems transporting calcium are present on the inner nuclear membrane. The putative channels would allow the entry of calcium into the nucleoplasm whereas the elusive transporting system(s) would contribute to replenish the nuclear stores. The simple proposed model is versatile enough to explain and predict autonomous changes in free calcium in the nucleoplasm of isolated plant nuclei.     

Sphingolipid metabolites selectively elicit increases in nuclear calcium concentration in cell suspension cultures and in isolated nuclei of tobacco

Sphingolipids are known to interfere with calcium-based signalling pathways. Here we report that these compounds modulate nuclear calcium signalling in tobacco BY-2 cells. Nuclear protein kinase activity phosphorylated endogenous sphingoid long-chain bases (LCBs), suggesting that LCBs are actively metabolized in the nucleus of tobacco BY-2 cells. The Delta4-unsaturated LCB D-erythro-sphingosine and the saturated LCB D-ribo-phytosphingosine elicited increases in free calcium in the nucleus in a dose-dependent and structure-related manner. However, neither sphingosine-1-phosphate nor C2-ceramide was able to stimulate nuclear calcium changes. N-,N-Dimethyl-D-erythro-sphingosine, a structural analogue of D-erythro-sphingosine, was the most efficient LCB so far tested in eliciting nuclear calcium changes both in intact tobacco BY-2 cells and in isolated nuclei. TRP channel inhibitors prevent the effect of DMS, suggesting that LCBs may activate TRP-like channels located on the inner nuclear membrane Collectively, the obtained data show that nuclei respond to LCBs on their own independently of the cytosolic compartment.     

Advances of calcium signals involved in plant anti-drought

Considerable progresses have taken place, both in the methodology available to study changes in intracellular cytosolic calcium and in our understanding of calcium signaling cascades, but how calcium signals function in plant drought resistance is questionable. In plant cells, calcium plays roles as a second messenger coupling a wide range of extracellular stimuli with intracellular responses. Different extracellular stimuli trigger specific calcium signatures: dynamics, amplitude and duration of calcium transients specify the nature, implication and intensity of stimuli. Calcium-binding proteins (sensors) play a critical role in decoding calcium signatures and transducing signals by activating specific targets and corresponding metabolic pathways. Calmodulin is a calcium sensor known to regulate the activity of many mammalian proteins, whose targets in plants are now being identified. Higher plants possess a rapidly growing list of calmodulin targets with a variety of cellular functions. Nevertheless, many targets appear to be unique to higher plants and remain characterized, calling for a concerted effort to elucidate their functions. To date, three major classes of plant calcium signals, including calcium permeable ion channels, Ca(2+)/H(+) antiporters and Ca(2+)-ATPases, have been responsible for drought-stress signal transduction. This review summarizes the current knowledge of calcium signals involved in plant anti-drought and plant water use efficiency (WUE) and presents suggestions for future focus of study.     

Afferent signals from pigeon extraocular muscles modify the vestibular responses of units in the abducens nucleus.

Although the extraocular muscles contain stretch receptors it is generally believed that their afferents exert no influence on the control of eye movement. However, we have shown previously that these afferent signals reach various brainstem centres concerned with eye movement, notably the vestibular nuclei, and that the decerebrate pigeon is a favourable preparation in which to study their effects. If the extraocular muscle afferents do influence oculomotor control from moment-to-moment they should exert a demonstrable effect on the oculomotor nuclei. We now present evidence that extraocular muscle afferent signals do, indeed, alter the responses of units in an oculomotor nucleus (the abducens, VI nerve nucleus, which supplies the lateral rectus muscle) to horizontal, vestibular stimulation induced by sinusoidal oscillation of the bird. Such stimuli evoke a vestibulo-ocular reflex in the intact bird. The extraocular stretch receptors were activated by passive eye movement within the pigeon's saccadic range; such movements modified the vestibular responses of all 19 units studied which were all, histologically, in the abducens nucleus. The magnitude of the effects, purely inhibitory in 15 units, depended both on the amplitude and the velocity of the eye movement and most units showed selectivity for particular combinations of plane (e.g. horizontal versus vertical) and direction (e.g. rostral versus caudal) of eye movement. The results show that an afferent signal from the extraocular muscles influences vestibularly driven activity in the abducens nucleus to which it carries information related to amplitude, velocity, plane and direction of eye movement in the saccadic range. They thus strongly support the view that extraocular afferent signals are involved in the control of eye movement.     

Specific binding of phorbol esters to nuclei of human promyelocytic leukemia cells

In this report, we demonstrate that HL-60 nuclei isolated in calcium but not EGTA containing buffers specifically bind PE and express approximately 37,000 receptor sites/nucleus. Nuclear phorbol ester binding is lost by isolation in the absence of calcium, but can be repleted by the addition of partially purified protein kinase C and calcium. When HL-60 cells are treated with bryostatin 1, a compound which activates protein kinase C in a similar fashion to phorbol esters but does not induce differentiation of HL-60 cells, and nuclei are isolated in the presence of EGTA, these nuclei continue to bind phorbol esters. These experiments suggest that HL-60 nuclei bind PE in vitro, and that compounds that activate protein kinase C may increase nuclear binding of PE in situ.     

Phospholipases in the nucleus

There is a well established role for various phospholipases involved in the production of intracellular signals at the plasma membrane. In contrast much less is known of their role in other intracellular compartments, however, emerging evidence would suggest that some of these enzymes are also involved in the production of signals within the nucleus. Translocation to and activation of protein kinase C (PKC) within the nucleus has been suggested to be important in a number of diverse cellular processes suggesting the requirement for the intranuclear production of diacylglycerol (DAG), a known physiological activator of this enzyme. As the activation of a number of phospholipases leads to the production of DAG this review will consider the notion that these enzymes are present within the nucleus and that their activities can be stimulated to produce this important regulator of PKC.     

Bioengineering plant resistance to abiotic stresses by the global calcium signal system

Considerable progresses have taken place both in the methodology available to study changes in intracellular cytosolic calcium and in our understanding of calcium signaling cascades. It is generally accepted that the global calcium signal system functions importantly in coping with plant abiotic stresses, especially drought stress, which has been proved further by the recent transgenic and molecular breeding reports under soil water deficits. In plant cells, calcium plays roles as a universal transducer coupling a wide range of extracellular stimuli with intracellular responses. Different extracellular stimuli trigger specific calcium signatures: dynamics, amplitude and duration of calcium transients specify the nature, implication and intensity of stimuli. Calcium-binding proteins (sensors) play a critical role in decoding calcium signatures and transducing signals by activating specific targets and corresponding metabolic pathways. Calmodulin (CAM) is a calcium sensor known to regulate the activity of many mammalian proteins, whose targets in plants are now being identified. Higher plants possess a rapidly growing list of CAM targets with a variety of cellular functions. Nevertheless, many targets appear to be unique to higher plant cells and remain characterized, calling for a concerted effort from plant and animal scientists to elucidate their functions. To date, three major classes of plant calcium signals encoding elements in the calcium signal system, including calcium-permeable ion channels,Ca(2)+/ H(+) antiporters and Ca(2)+-ATPases, are responsible for drought stress signal transduction directly or indirectly. This review summarizes the current knowledge of calcium signals involved in plant abiotic stresses and presents suggestions for future focus areas of study.     

Ionic Permeability on Isolated Mouse Liver Nuclei: Influence of ATP and Ca2+

Patch-clamp experiments on isolated nuclei revealed the existence of ionic channels on the nuclear envelope, but their exact localization and function are still unknown. Recent studies have demonstrated that ATP and calcium ions play an important role in nucleocytoplasmic protein traffic. ATP is essential to allow big molecules in and out of the nucleus. However, a cytoplasmic rise of calcium ions above 300 nm decreases both ATP-dependent transport and passive diffusion through the nuclear envelope. The use of isolated nuclei placed in a saline solution provides the possibility for testing only the compounds added in the bath or in the recording pipette. In the present study, we show that ATP is responsible for an increase of nuclear ionic permeability on isolated nuclei. This result not only confirms data previously reported in in situ nuclei, but also suggests that ATP is directly involved in the modulation of passive ionic permeability. In these particular experimental conditions, calcium ions decrease the channel current starting from a concentration of 1 μm. The parallelism in the modulation action of ATP and Ca⁺⁺ between nuclear pores and ionic channels present on the nuclear envelope contributes to the support of the idea that an ionic pathway is associated with the pore complex. Received: 5 September 1996/Revised: 13 January 1997     

Calcium microdomains and cell cycle control

The cell division cycle comprises successive rounds of genome replication and segregation that are never error-free. A complex signalling network chaperones cell cycle events to ensure that cell cycle progression does not occur until any errors detected are put right. The signalling network consists of cell cycle control proteins that are phosphorylated and dephosphorylated, synthesized and degraded interactively to generate a set of sensors and molecular switches that are thrown at appropriate times to permit or trigger cell cycle progression. In early embryos, discrete calcium signals have been shown to be a key component of the molecular switch mechanism. In somatic cells in contrast, the participation of calcium signals in cell cycle control is far from clear. Recent experiments in syncytial Drosophila embryos have shown that localised calcium signals in the nucleus and mitotic spindle can be detected. It appears that the nucleus comprises a calcium signalling microdomain bounded by endoplasmic reticulum that isolates the nucleus and spindle. These findings offer a possible explanation for the apparent absence of calcium signals in somatic cells during mitosis.     

Neural coding in the chick cochlear nucleus

Physiological recordings were made from single units in the two divisions of the chick cochlear nucleus-nucleus angularis (NA) and nucleus magnocellularis (NM). Sound evoked responses were obtained in an effort to quantify functional differences between the two nuclei. In particular, it was of interest to determine if nucleus angularis and magnocellularis code for separate features of sound stimuli, such as temporal and intensity information. The principal findings are: 1. Spontaneous activity patterns in the two nuclei are very different. Neurons in nucleus angularis tend to have low spontaneous discharge rates while magnocellular units have high levels of spontaneous firing. 2. Frequency tuning curves recorded in both nuclei are similar in form, although the best thresholds of NA units are about 10 dB more sensitive than their NM counterparts across the entire frequency range. A wide spread of neural thresholds is evident in both NA and NM. 3. Large driven increases in discharge rate are seen in both NA and NM. Rate intensity functions from NM units are all monotonic, while a substantial percentage (22%) of NA units respond to increased sound level in a nonmonotonic fashion. 4. Most NA units with characteristic frequencies (CF) above 1000 Hz respond to sound stimuli at CF as 'choppers', while units with CF's below 1000 Hz are 'primary-like'. Several 'onset' units are also seen in NA. In contrast, all NM units show 'primary-like' response. 5. Units in both nuclei with CF's below 1000 Hz show strong neural phase-locking to stimuli at their CF. Above 1000 Hz, few NA units are phase-locked, while phase-locking in NM extends to 2000 Hz. 6. These results are discussed with reference to the hypothesis that NM initiates a neural pathway which codes temporal information while NA is involved primarily with intensity coding, similar in principle to the segregation of function seen in the cochlear nucleus of the barn owl (Sullivan and Konishi 1984).     

pH Change in Culture Medium Induces Reorganization of Intranuclear Actin in Two-Cell Mouse Embryos

Actin is a permanent component of the cell nucleus involved in many nuclear processes. However, some nuclear functions of actin remain insufficiently explored. The role played by various extracellular stimuli in regulation of nuclear actin still remains enigmatic. Deviation of basic parameters of culture medium from optimal values is a member of the group of extracellular stimuli that are very important for mammalian embryos cultured in vitro. Change in culture medium pH from the level optimal for embryo homeostasis is one such signals. The purpose of this study was to investigate the intranuclear actin distribution in nuclei of two-cell mouse embryos under stress conditions induced by changes in extracellular pH. The pattern of actin localization has been tracked after short-term culturing of the embryos at optimal (pH 7.2), increased (pH 7.8), or decreased (pH 6.5) pH conditions. Analysis was carried out with confocal microscopy using methods of direct fluorescent and indirect immunofluorescent identification of actin. It has been shown that the change of culture medium pH from the optimum value is the signal that alters intranuclear actin distribution in nuclei of the embryonic cells. Culture of two-cell mouse embryos in suboptimal pH conditions (pH 6.5 and pH 7.8) induced alterations in the intranuclear actin localization, which, in particular, were expressed in accumulation of monomeric actin and the appearance of phalloidin-stainable actin in the nuclei. These changes, in our opinion, show some signs of similarity with stress-induced changes in nuclear-actin distribution, which, as has been reported earlier by a number of researchers, have been observed in the nuclei of somatic cells.     

Calcium signaling in plant cell organelles delimited by a double membrane

Increases in the concentration of free calcium in the cytosol are one of the general events that relay an external stimulus to the internal cellular machinery and allow eukaryotic organisms, including plants, to mount a specific biological response. Different lines of evidence have shown that other intracellular organelles contribute to the regulation of free calcium homeostasis in the cytosol. The vacuoles, the endoplasmic reticulum and the cell wall constitute storage compartments for mobilizable calcium. In contrast, the role of organelles surrounded by a double membrane (e.g. mitochondria, chloroplasts and nuclei) is more complex. Here, we review experimental data showing that these organelles harbor calcium-dependent biological processes. Mitochondria, chloroplasts as well as nuclei are equipped to generate calcium signal on their own. Changes in free calcium in a given organelle may also favor the relocalization of proteins and regulatory components and therefore have a profound influence on the integrated functioning of the cell. Studying, in time and space, the dynamics of different components of calcium signaling pathway will certainly give clues to understand the extraordinary flexibility of plants to respond to stimuli and mount adaptive responses. The availability of technical and biological resources should allow breaking new grounds by unveiling the contribution of signaling networks in integrative plant biology.     

Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)_n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.     

The development of the static vestibulo-ocular reflex in the Southern Clawed Toad,Xenopus laevis

The static vestibulo-ocular reflex was investigated in tadpoles at different times following unilateral destruction of the labyrinth during the period of early organogenesis and premetamorphosis. Balance compensation is completed after a few weeks, while gain compensation only occurs partially (Figs. 2-4). Tadpoles hemilabyrinthectomized in the age of 2.5 days (stage 38) develop no vestibular nuclei on their lesioned side, while tadpoles operated later in their life, possess these nuclei (Figs. 5, 6) even if they were not detectable at the operation day (Fig. 7). For their dorsal vestibular nucleus (DVN), the number of neurons is usually larger on the intact than on the lesioned side; while for the ventral vestibular nucleus (VVN), there is either numerical symmetry or a transient decrease of cell number on the intact side (Fig. 5). The results demonstrate that vestibular compensation occurs even if vestibular nuclei have developed only on one side, i.e. the vestibular commissure is not a prerequisite for a successful compensation process. It is discussed whether the use of extra-vestibular error signals for balance but not for gain compensation may cause the differences in time courses of both compensation processes.     

Plant protein kinases stimulated by calcium

Calcium signals play an important role in many aspects of plant growth and development, including plant response to biotic and abiotic stress. The stimulus characteristic intracellular Ca2+ signals are generated in plant cells by a variety of stimuli, including changes in environmental conditions, interaction with microbes and growth and development processes. Cytoplasmatic calcium brings about responses by interacting with target proteins, like calcium-dependent kinases. In plant there are at least five classes of protein kinases (CDPK, CRK, CCaMK, CaMK and SnRK3), which activity is regulated by calcium ions. In this article the structure, regulation and function of calcium stimulated protein kinases are briefly reviewed.