Control of time-dependent biological processes by temporally patterned input

Temporal patterning of biological variables, in the form of oscillations and rhythms on many time scales, is ubiquitous. Altering the temporal pattern of an input variable greatly affects the output of many biological processes. We develop here a conceptual framework for a quantitative understanding of such pattern dependence, focusing particularly on nonlinear, saturable, time-dependent processes that abound in biophysics, biochemistry, and physiology. We show theoretically that pattern dependence is governed by the nonlinearity of the input–output transformation as well as its time constant. As a result, only patterns on certain time scales permit the expression of pattern dependence, and processes with different time constants can respond preferentially to different patterns. This has implications for temporal coding and decoding, and allows differential control of processes through pattern. We show how pattern dependence can be quantitatively predicted using only information from steady, unpatterned input. To apply our ideas, we analyze, in an experimental example, how muscle contraction depends on the pattern of motorneuron firing.

A modifier screen in the eye reveals control genes for Krüppel activity in the Drosophila embryo

Irregular facets (If) is a dominant mutation of Drosophila that results in small eyes with fused ommatidia. Previous results showed that the gene Krüppel (Kr), which is best known for its early segmentation function, is expressed ectopically in If mutant eye discs. However, it was not known whether ectopic Kr activity is either the cause or the result of the If mutation. Here, we show that If is a gain-of-function allele of Kr. We then used the If mutation in a genetic screen to identify dominant enhancers and suppressors of Kr activity on the third chromosome. Of 30 identified Kr-interacting loci, two were cloned, and we examined whether they also represent components of a natural Kr-dependent developmental pathway of the embryo. We show that the two genes, eyelid (eld) and extramacrochaetae (emc), which encode a Bright family-type DNA binding protein and a helix-loop-helix factor, respectively, are necessary to achieve the singling-out of a unique Kr-expressing cell during the development of the Malpighian tubules, the excretory organs of the fly. The results indicate that the Kr gain-of-function mutation If provides a tool to identify genes that are active during eye development and that a number of them function also in the control of Kr-dependent developmental processes.

Ephrin-dependent growth and pruning of hippocampal axons

Neuronal connections are arranged topographically such that the spatial organization of neurons is preserved by their termini in the targets. During the development of topographic projections, axons initially explore areas much wider than the final targets, and mistargeted axons are pruned later. The molecules regulating these processes are not known. We report here that the ligands of the Eph family tyrosine kinase receptors may regulate both the initial outgrowth and the subsequent pruning of axons. In the presence of ephrins, the outgrowth and branching of the receptor-positive hippocampal axons are enhanced. However, these axons are induced later to degenerate. These observations suggest that the ephrins and their receptors may regulate topographic map formation by stimulating axonal arborization and by pruning mistargeted axons.

Caspase-dependent activation of cyclin-dependent kinases during Fas-induced apoptosis in Jurkat cells

The activation of cyclin-dependent kinases (cdks) has been implicated in apoptosis induced by various stimuli. We find that the Fas-induced activation of cdc2 and cdk2 in Jurkat cells is not dependent on protein synthesis, which is shut down very early during apoptosis before caspase-3 activation. Instead, activation of these kinases seems to result from both a rapid cleavage of Wee1 (an inhibitory kinase of cdc2 and cdk2) and inactivation of anaphase-promoting complex (the specific system for cyclin degradation), in which CDC27 homolog is cleaved during apoptosis. Both Wee1 and CDC27 are shown to be substrates of the caspase-3-like protease. Although cdk activities are elevated during Fas-induced apoptosis in Jurkat cells, general activation of the mitotic processes does not occur. Our results do not support the idea that apoptosis is simply an aberrant mitosis but, instead, suggest that a subset of mitotic mechanisms plays an important role in apoptosis through elevated cdk activities.

Crossover isomer bias is the primary sequence-dependent property of immobilized Holliday junctions

Recombination of genes is essential to the evolution of genetic diversity, the segregation of chromosomes during cell division, and certain DNA repair processes. The Holliday junction, a four-arm, four-strand branched DNA crossover structure, is formed as a transient intermediate during genetic recombination and repair processes in the cell. The recognition and subsequent resolution of Holliday junctions into parental or recombined products appear to be critically dependent on their three-dimensional structure. Complementary NMR and time-resolved fluorescence resonance energy transfer experiments on immobilized four-arm DNA junctions reported here indicate that the Holliday junction cannot be viewed as a static structure but rather as an equilibrium mixture of two conformational isomers. Furthermore, the distribution between the two possible crossover isomers was found to depend on the sequence in a manner that was not anticipated on the basis of previous low-resolution experiments.

Activity-dependent regulation of synaptic clustering in a hippocampal culture system

Currently, there is a limited understanding of the factors that influence the localization and density of individual synapses in the central nervous system. Here we have studied the effects of activity on synapse formation between hippocampal dentate granule cells and CA3 pyramidal neurons in culture, taking advantage of FM1–43 as a fluorescent marker of synaptic boutons. We observed an early tendency for synapses to group together, quickly followed by the appearance of synaptic clusters on dendritic processes. These events were strongly influenced by N-methyl-d-aspartic acid receptor- and cyclic AMP-dependent signaling. The microstructure and localization of the synaptic clusters resembled that found in hippocampus, at mossy fiber synapses of stratum lucidum. Activity-dependent clustering of synapses represents a means for synaptic targeting that might contribute to synaptic organization in the brain.

Thermus thermophilus: A link in evolution of the tRNA-dependent amino acid amidation pathways

Thermus thermophilus possesses an aspartyl-tRNA synthetase (AspRS2) able to aspartylate efficiently tRNAAsp and tRNAAsn. Aspartate mischarged on tRNAAsn then is converted into asparagine by an ω amidase that differs structurally from all known asparagine synthetases. However, aspartate is not misincorporated into proteins because the binding capacity of aminoacylated tRNAAsn to elongation factor Tu is only conferred by conversion of aspartate into asparagine. T. thermophilus additionally contains a second aspartyl-tRNA synthetase (AspRS1) able to aspartylate tRNAAsp and an asparaginyl-tRNA synthetase able to charge tRNAAsn with free asparagine, although the organism does not contain a tRNA-independent asparagine synthetase. In contrast to the duplicated pathway of tRNA asparaginylation, tRNA glutaminylation occurs in the thermophile via the usual pathway by using glutaminyl-tRNA synthetase and free glutamine synthesized by glutamine synthetase that is unique. T. thermophilus is able to ensure tRNA aminoacylation by alternative routes involving either the direct pathway or by conversion of amino acid mischarged on tRNA. These findings shed light on the interrelation between the tRNA-dependent and tRNA-independent pathways of amino acid amidation and on the processes involved in fidelity of the aminoacylation systems.

Go protein-dependent survival of primary accessory olfactory neurons

Extensive G protein-coupled receptor families in both the main and accessory olfactory systems have been implicated in axonal targeting, sensory function, and cell survival. Although sensory function seems to be mediated by G proteins, axonal guidance and cell survival may be G protein-independent processes. In the accessory olfactory system, the Go-containing neurons in the basal vomeronasal organ (VNO) project to the posterior accessory olfactory bulb (AOB), whereas more apically located VNO neurons contain Gi2 and project to the anterior AOB. Herein, we investigate the organization of the accessory olfactory system in mice with a targeted deletion in the Goα gene. The accessory olfactory system seems normal at birth; however, postnatally, the number of Go-receptor-containing VNO neurons decreases by half, and apoptotic neurons are detected. The axons of VNO neurons remain restricted to the posterior AOB. The posterior AOB is reduced in size but contains a synaptophysin-positive layer with the normal number of glomeruli. The posterior AOB has reduced mitral cell c-Fos immunoreactivity, consistent with decreased sensory activation of Go protein-coupled VNO receptor neurons. Thus, in the accessory olfactory system, receptor-coupled G proteins are required for cell survival.

The polo-box-dependent induction of ectopic septal structures by a mammalian polo kinase, Plk, in Saccharomyces cerevisiae

Members of the polo subfamily of protein kinases play pivotal roles in cell-cycle control and proliferation. In addition to a high degree of sequence similarity in the kinase domain, polo kinases contain a strikingly conserved motif termed “polo-box” in the noncatalytic C-terminal domain. We have previously shown that the mammalian polo-like kinase Plk is a functional homolog of Saccharomyces cerevisiae Cdc5. Here, we show that, in a polo-box- and kinase activity-dependent manner, ectopic expression of Plk in budding yeast can induce a class of cells with abnormally elongated buds. In addition to localization at spindle poles and cytokinetic neck filaments, Plk induces and localizes to ectopic septin ring structures within the elongated buds. In contrast, mutations in the polo-box abolish both localization to, and induction of, septal structures. Consistent with the polo-box-dependent subcellular localization, the C-terminal domain of Plk, but not its polo-box mutant, is sufficient for subcellular localization. Our data suggest that Plk may contribute a signal to initiate or promote cytokinetic event(s) and that an intact polo-box is required for regulation of these cellular processes.

P element insertion-dependent gene activation in the Drosophila eye

Insights into the function of a gene can be gained in multiple ways, including loss-of-function phenotype, sequence similarity, expression pattern, and by the consequences of its misexpression. Analysis of the phenotypes produced by expression of a gene at an abnormal time, place, or level may provide clues to a gene’s function when other approaches are not illuminating. Here we report that an eye-specific, enhancer–promoter present in the P element expression vector pGMR is able to drive high level expression in the eye of genes near the site of P element insertion. Cell fate determination, differentiation, proliferation, and death are essential for normal eye development. Thus the ability to carry out eye-specific misexpression of a significant fraction of genes in the genome, given the dispensability of the eye for viability and fertility of the adult, should provide a powerful approach for identifying regulators of these processes. To test this idea we carried out two overexpression screens for genes that function to regulate cell death. We screened for insertion-dependent dominant phenotypes in a wild-type background, and for dominant modifiers of a reaper overexpression-induced small eye phenotype. Multiple chromosomal loci were identified, including an insertion 5′ to hid, a potent inducer of apoptosis, and insertions 5′ to DIAP1, a cell death suppressor. To facilitate the cloning of genes near the P element insertion new misexpression vectors were created. A screen with one of these vectors identified eagle as a suppressor of a rough eye phenotype associated with overexpression of an activated Ras1 gene.

Dual pathways for regulation of root branching by nitrate

Root development is extremely sensitive to variations in nutrient supply, but the mechanisms are poorly understood. We have investigated the processes by which nitrate (NO3−), depending on its availability and distribution, can have both positive and negative effects on the development and growth of lateral roots. When Arabidopsis roots were exposed to a locally concentrated supply of NO3− there was no increase in lateral root numbers within the NO3−-rich zone, but there was a localized 2-fold increase in the mean rate of lateral root elongation, which was attributable to a corresponding increase in the rate of cell production in the lateral root meristem. Localized applications of other N sources did not stimulate lateral root elongation, consistent with previous evidence that the NO3− ion is acting as a signal rather than a nutrient. The axr4 auxin-resistant mutant was insensitive to the stimulatory effect of NO3−, suggesting an overlap between the NO3− and auxin response pathways. High rates of NO3− supply to the roots had a systemic inhibitory effect on lateral root development that acted specifically at the stage when the laterals had just emerged from the primary root, apparently delaying final activation of the lateral root meristem. A nitrate reductase-deficient mutant showed increased sensitivity to this systemic inhibitory effect, suggesting that tissue NO3− levels may play a role in generating the inhibitory signal. We present a model in which root branching is modulated by opposing signals from the plant’s internal N status and the external supply of NO3−.

Microtubule self-organization is gravity-dependent

Although weightlessness is known to affect living cells, the manner by which this occurs is unknown. Some reaction-diffusion processes have been theoretically predicted as being gravity-dependent. Microtubules, a major constituent of the cellular cytoskeleton, self-organize in vitro by way of reaction-diffusion processes. To investigate how self-organization depends on gravity, microtubules were assembled under low gravity conditions produced during space flight. Contrary to the samples formed on an in-flight 1 × g centrifuge, the samples prepared in microgravity showed almost no self-organization and were locally disordered.

Replication-dependent early meiotic requirement for Spo11 and Rad50

Spo11 and the Rad50-Mre11 complex have been indirectly implicated in processes associated with DNA replication. These proteins also have been shown to have early meiotic roles essential for the formation of a programmed DNA double-strand break known in Saccharomyces cerevisiae to initiate meiotic recombination. In both S. cerevisiae and the basidiomycete Coprinus cinereus, spo11 and rad50 mutants are defective in chromosome synapsis during meiosis. Here we demonstrate that a partial restoration of synapsis occurs in C. cinereus spo11 and rad50 mutants if premeiotic DNA replication is prevented. Double mutants were constructed with spo11–1 or rad50–4 and another mutant, spo22–1, which does not undergo premeiotic DNA replication. In both cases, we observed an increase in the percentage of nuclei containing synaptonemal complex (SC) structures, with concomitant decreases in the percentage of nuclei containing axial elements (AE) only or no structures. Both types of double mutants demonstrated significant increases in the average numbers of AE and SC, although SC-containing nuclei did not on average contain more AE than did nuclei showing no synapsis. Our results show that Spo11-induced recombination is not absolutely required for synapsis in C. cinereus, and that the early meiotic role of both Spo11 and Rad50 in SC formation partially depends on premeiotic S phase. This dependency likely reflects either a requirement for these proteins imposed by the premeiotic replication process itself or a requirement for these proteins in synapsis when a sister chromatid (the outcome of DNA replication) is present.

Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells

The pancreatic acinar cell produces powerful digestive enzymes packaged in zymogen granules in the apical pole. Ca2+ signals elicited by acetylcholine or cholecystokinin (CCK) initiate enzyme secretion by exocytosis through the apical membrane. Intracellular enzyme activation is normally kept to a minimum, but in the often-fatal human disease acute pancreatitis, autodigestion occurs. How the enzymes become inappropriately activated is unknown. We monitored the cytosolic Ca2+ concentration ([Ca2+]i), intracellular trypsin activation, and its localization in isolated living cells with specific fluorescent probes and studied intracellular vacuole formation by electron microscopy as well as quantitative image analysis (light microscopy). A physiological CCK level (10 pM) eliciting regular Ca2+ spiking did not evoke intracellular trypsin activation or vacuole formation. However, stimulation with 10 nM CCK, evoking a sustained rise in [Ca2+]i, induced pronounced trypsin activation and extensive vacuole formation, both localized in the apical pole. Both processes were abolished by preventing abnormal [Ca2+]i elevation, either by preincubation with the specific Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N-N′,N′-tetraacetic acid (BAPTA) or by removal of external Ca2+. CCK hyperstimulation evokes intracellular trypsin activation and vacuole formation in the apical granular pole. Both of these processes are mediated by an abnormal sustained rise in [Ca2+]i.

UV-damage-mediated induction of homologous recombination in Arabidopsis is dependent on photosynthetically active radiation

Plants are continuously subjected to UV-B radiation (UV-B; 280–320 nm) as a component of sunlight causing damage to the genome. For elimination of DNA damage, a set of repair mechanisms, mainly photoreactivation, excision, and recombination repair, has evolved. Whereas photoreactivation and excision repair have been intensely studied during the last few years, recombination repair, its regulation, and its interrelationship with photoreactivation in response to UV-B-induced DNA damage is still poorly understood. In this study, we analyzed somatic homologous recombination in a transgenic Arabidopsis line carrying a β-glucuronidase gene as a recombination marker and in offsprings of crosses of this line with a photolyase deficient uvr2–1 mutant. UV-B radiation stimulated recombination frequencies in a dose-dependent manner correlating linearly with cyclobutane pyrimidine dimer (CPD) levels. Genetic deficiency for CPD-specific photoreactivation resulted in a drastic increase of recombination events, indicating that homologous recombination might be directly involved in eliminating CPD damage. UV-B irradiation stimulated recombination mainly in the presence of photosynthetic active radiation (400–700 nm) irrespective of photolyase activities. Our results suggest that UV-B-induced recombination processes may depend on energy supply derived from photosynthesis.