Coilin Shuttles between the Nucleus and Cytoplasm In Xenopus Oocytes

Coiled bodies are discrete nuclear organelles often identified by the marker protein p80-coilin. Because coilin is not detected in the cytoplasm by immunofluorescence and Western blotting, it has been considered an exclusively nuclear protein. In the Xenopus germinal vesicle (GV), most coilin actually resides in the nucleoplasm, although it is highly concentrated in 50–100 coiled bodies. When affinity-purified anti-coilin antibodies were injected into the cytoplasm of oocytes, they could be detected in coiled bodies within 2–3 h. Coiled bodies were intensely labeled after 18 h, whereas other nuclear organelles remained negative. Because the nuclear envelope does not allow passive diffusion of immunoglobulins, this observation suggests that anti-coilin antibodies are imported into the nucleus as an antigen–antibody complex with coilin. Newly synthesized coilin is not required, because cycloheximide had no effect on nuclear import and subsequent targeting of the antibodies. Additional experiments with myc-tagged coilin and myc-tagged pyruvate kinase confirmed that coilin is a shuttling protein. The shuttling of Nopp140, NO38/B23, and nucleolin was easily demonstrated by the targeting of their respective antibodies to the nucleoli, whereas anti-SC35 did not enter the germinal vesicle. We suggest that coilin, perhaps in association with Nopp140, may function as part of a transport system between the cytoplasm and the coiled bodies.

Cytoplasmically sequestered wild-type p53 protein in neuroblastoma is relocated to the nucleus by a C-terminal peptide

Cytoplasmic sequestration of wild-type p53 protein occurs in a subset of primary human tumors including breast cancer, colon cancer, and neuroblastoma (NB). The sequestered p53 localizes to punctate cytoplasmic structures that represent large protein aggregates. One functional consequence of this blocked nuclear access is impairment of the p53-mediated G1 checkpoint after DNA damage. Here we show that cytoplasmic p53 from NB cells is incompetent for specific DNA binding, probably due to its sequestration. Importantly, the C-terminal domain of sequestered p53 is masked, as indicated by the failure of a C-terminally directed antibody to detect p53 in these structures. To determine (i) which domain of p53 is involved in the aggregation and (ii) whether this phenotype is potentially reversible, we generated stable NB sublines that coexpress the soluble C-terminal mouse p53 peptide DD1 (amino acids 302–390). A dramatic phenotypic reversion occurred in five of five lines. The presence of DD1 blocked the sequestration of wild-type p53 and relocated it to the nucleus, where it accumulated. The nuclear translocation is due to shuttling of wild-type p53 by heteroligomerization to DD1, as shown by coimmunoprecipitation. As expected, the nuclear heterocomplexes were functionally inactive, since DD1 is a dominant negative inhibitor of wild-type p53. In summary, we show that nuclear access of p53 can be restored in NB cells.

Multiple independent transpositions of mitochondrial DNA control region sequences to the nucleus

Transpositions of mtDNA sequences to the nuclear genome have been documented in a wide variety of individual taxa, but little is known about their taxonomic frequency or patterns of variation. We provide evidence of nuclear sequences homologous to the mtDNA control region in seven species of diving ducks (tribe Aythyini). Phylogenetic analysis places each nuclear sequence as a close relative of the mtDNA haplotypes of the specie(s) in which it occurs, indicating that they derive from six independent transposition events, all occurring within the last ≈1.5 million years. Relative-rate tests and comparison of intraspecific variation in nuclear and mtDNA sequences confirm the expectation of a greatly reduced rate of evolution in the nuclear copies. By representing mtDNA haplotypes from ancestral populations, nuclear insertions may be valuable in some phylogenetic analyses, but they also confound the accurate determination of mtDNA sequences. In particular, our data suggest that the presumably nonfunctional but more slowly evolving nuclear sequences often will not be identifiable by changes incompatible with function and may be preferentially amplified by PCR primers based on mtDNA sequences from related taxa.

Transition-state structure as a unifying basis in protein-folding mechanisms: Contact order, chain topology, stability, and the extended nucleus mechanism

I attempt to reconcile apparently conflicting factors and mechanisms that have been proposed to determine the rate constant for two-state folding of small proteins, on the basis of general features of the structures of transition states. Φ-Value analysis implies a transition state for folding that resembles an expanded and distorted native structure, which is built around an extended nucleus. The nucleus is composed predominantly of elements of partly or well-formed native secondary structure that are stabilized by local and long-range tertiary interactions. These long-range interactions give rise to connecting loops, frequently containing the native loops that are poorly structured. I derive an equation that relates differences in the contact order of a protein to changes in the length of linking loops, which, in turn, is directly related to the unfavorable free energy of the loops in the transition state. Kinetic data on loop extension mutants of CI2 and α-spectrin SH3 domain fit the equation qualitatively. The rate of folding depends primarily on the interactions that directly stabilize the nucleus, especially those in native-like secondary structure and those resulting from the entropy loss from the connecting loops, which vary with contact order. This partitioning of energy accounts for the success of some algorithms that predict folding rates, because they use these principles either explicitly or implicitly. The extended nucleus model thus unifies the observations of rate depending on both stability and topology.

Retinotopic mapping of lateral geniculate nucleus in humans using functional magnetic resonance imaging

Subcortical nuclei in the thalamus, which play an important role in many functions of the human brain, provide challenging targets for functional mapping with neuroimaging techniques because of their small sizes and deep locations. In this study, we explore the capability of high-resolution functional magnetic resonance imaging at 4 Tesla for mapping the retinotopic organization in the lateral geniculate nucleus (LGN). Our results show that the hemifield visual stimulation only activates LGN in the contralateral hemisphere, and the lower-field and upper-field visual stimulations activate the superior and inferior portion of LGN, respectively. These results reveal a similar retinotopic organization between the human and nonhuman primate LGN and between LGN and the primary visual cortex. We conclude that high-resolution functional magnetic resonance imaging is capable of functional mapping of suborganizations in small nuclei together with cortical activation. This will have an impact for studying the thalamocortical networks in the human brain.

Reversible inactivation of the nucleus basalis magnocellularis induces disruption of cortical acetylcholine release and acquisition, but not retrieval, of aversive memories

The basal forebrain complex, which includes the nucleus basalis magnocellularis (NBM), provides widespread cholinergic and γ-aminobutyric acid-containing projections throughout the brain, including the insular and pyriform cortices. A number of studies have implicated the cholinergic neurons in the mediation of learning and memory processes. However, the role of basal forebrain activity in information retrieval mechanisms is less known. The aim of the present study is to evaluate the effects of reversible inactivation of the NBM by tetrodotoxin (TTX, a voltage-sensitive sodium channel blocker) during the acquisition and retrieval of conditioned taste aversion (CTA) and to measure acetylcholine (ACh) release during TTX inactivation in the insular cortex, by means of the microdialysis technique in free-moving rats. Bilateral infusion of TTX in the NBM was performed 30 min before the presentation of gustative stimuli, in either the CTA acquisition trial or retrieval trial. At the same time, levels of extracellular ACh release were measured in the insular cortex. The behavioral results showed significant impairment in CTA acquisition when the TTX was infused in the NBM, whereas retrieval was not affected when the treatment was given during the test trial. Biochemical results showed that TTX infusion into the NBM produced a marked decrease in cortical ACh release as compared with the controls during consumption of saccharin in the acquisition trial. Depleted ACh levels were found during the test trial in all groups except in the group that received TTX during acquisition. These results suggest a cholinergic-dependent process during acquisition, but not during memory retrieval, and that NBM-mediated cholinergic cortical release may play an important role in early stages of learning, but not during recall of aversive memories.

Multi-responsiveness of single anterior pituitary cells to hypothalamic-releasing hormones: A cellular basis for paradoxical secretion

The classic view for hypothalamic regulation of anterior pituitary (AP) hormone secretion holds that release of each AP hormone is controlled specifically by a corresponding hypothalamic-releasing hormone (HRH). In this scenario, binding of a given HRH (thyrotropin-, growth hormone-, corticotropin-, and luteinizing hormone-releasing hormones) to specific receptors in its target cell increases the concentration of cytosolic Ca2+ ([Ca2+]i), thereby selectively stimulating the release of the appropriate hormone. However, “paradoxical” responses of AP cells to the four well-established HRHs have been observed repeatedly with both in vivo and in vitro systems, raising the possibility of functional overlap between the different AP cell types. To explore this possibility, we evaluated the effects of HRHs on [Ca2+]i in single AP cells identified immunocytochemically by the hormone they stored. We found that each of the five major AP cell types contained discrete subpopulations that were able to respond to several HRHs. The relative abundance of these multi-responsive cells was 59% for lactotropes, 33% for thyrotropes, and in the range of 47–55% for gonadotropes, corticotropes, and somatotropes. Analysis of prolactin release from single living cells revealed that each of the four HRHs tested were able to induce hormone release from a discrete lactotrope subpopulation, the size of which corresponded closely to that in which [Ca2+]i changes were induced by the same secretagogues. When viewed as a whole, our diverse functional measurements of multi-responsiveness suggest that hypothalamic control of pituitary function is more complicated than previously envisioned. Moreover, they provide a cellular basis for the so-called “paradoxical” behavior of pituitary cells to hypothalamic hypophysiotropic agents.

Neuroimaging of cerebral activations and deactivations associated with hypercapnia and hunger for air

There are defined medullary, mesencephalic, hypothalamic, and thalamic functions in regulation of respiration, but knowledge of cortical control and the elements subserving the consciousness of breathlessness and air hunger is limited. In nine young adults, air hunger was produced acutely by CO2 inhalation. Comparisons were made with inhalation of a N2/O2 gas mixture with the same apparatus, and also with paced breathing, and with eyes closed rest. A network of activations in pons, midbrain (mesencephalic tegmentum, parabrachial nucleus, and periaqueductal gray), hypothalamus, limbic and paralimbic areas (amygdala and periamygdalar region) cingulate, parahippocampal and fusiform gyrus, and anterior insula were seen along with caudate nuclei and pulvinar activations. Strong deactivations were seen in dorsal cingulate, posterior cingulate, and prefrontal cortex. The striking response of limbic and paralimbic regions points to these structures having a singular role in the affective sequelae entrained by disturbance of basic respiratory control whereby a process of which we are normally unaware becomes a salient element of consciousness. These activations and deactivations include phylogenetically ancient areas of allocortex and transitional cortex that together with the amygdalar/periamygdalar region may subserve functions of emotional representation and regulation of breathing.

Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction

A plastid-derived signal plays an important role in the coordinated expression of both nuclear- and chloroplast-localized genes that encode photosynthesis-related proteins. Arabidopsis GUN (genomes uncoupled) loci have been identified as components of plastid-to-nucleus signal transduction. Unlike wild-type plants, gun mutants have nuclear Lhcb1 expression in the absence of chloroplast development. We observed a synergistic phenotype in some gun double-mutant combinations, suggesting there are at least two independent pathways in plastid-to-nucleus signal transduction. There is a reduction of chlorophyll accumulation in gun4 and gun5 mutant plants, and a gun4gun5 double mutant shows an albino phenotype. We cloned the GUN5 gene, which encodes the ChlH subunit of Mg-chelatase. We also show that gun2 and gun3 are alleles of the known photomorphogenic mutants, hy1 and hy2, which are required for phytochromobilin synthesis from heme. These findings suggest that certain perturbations of the tetrapyrrole biosynthetic pathway generate a signal from chloroplasts that causes transcriptional repression of nuclear genes encoding plastid-localized proteins. The comparison of mutant phenotypes of gun5 and another Mg-chelatase subunit (ChlI) mutant suggests a specific function for ChlH protein in the plastid-signaling pathway.

Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors α- and γ-mediated gene expression via liver fatty acid binding protein: A signaling path to the nucleus

Peroxisome proliferator-activated receptor α (PPARα) is a key regulator of lipid homeostasis in hepatocytes and target for fatty acids and hypolipidemic drugs. How these signaling molecules reach the nuclear receptor is not known; however, similarities in ligand specificity suggest the liver fatty acid binding protein (L-FABP) as a possible candidate. In localization studies using laser-scanning microscopy, we show that L-FABP and PPARα colocalize in the nucleus of mouse primary hepatocytes. Furthermore, we demonstrate by pull-down assay and immunocoprecipitation that L-FABP interacts directly with PPARα. In a cell biological approach with the aid of a mammalian two-hybrid system, we provide evidence that L-FABP interacts with PPARα and PPARγ but not with PPARβ and retinoid X receptor-α by protein–protein contacts. In addition, we demonstrate that the observed interaction of both proteins is independent of ligand binding. Final and quantitative proof for L-FABP mediation was obtained in transactivation assays upon incubation of transiently and stably transfected HepG2 cells with saturated, monounsaturated, and polyunsaturated fatty acids as well as with hypolipidemic drugs. With all ligands applied, we observed strict correlation of PPARα and PPARγ transactivation with intracellular concentrations of L-FABP. This correlation constitutes a nucleus-directed signaling by fatty acids and hypolipidemic drugs where L-FABP acts as a cytosolic gateway for these PPARα and PPARγ agonists. Thus, L-FABP and the respective PPARs could serve as targets for nutrients and drugs to affect expression of PPAR-sensitive genes.

Refractoriness to melatonin occurs independently at multiple brain sites in Siberian hamsters

The mid-winter development of refractoriness to melatonin (Mel) triggers recrudescence of the atrophied reproductive apparatus of rodents. As a consequence, over-wintering animals become reproductively competent just before the onset of spring conditions favorable for breeding. The neural target tissues that cease to respond to winter Mel signals have not been identified. We now report that the suprachiasmatic nucleus of the hypothalamus, which contains the principal circadian clock, and the reuniens and paraventricular nuclei of the thalamus, each independently becomes refractory to melatonin. Small implants of Mel that were left in place for 40 wk and that act locally on these brain nuclei, induced testicular regression within 6 wk in male Siberian hamsters; 12 wk later Mel implants no longer suppressed reproduction and gonadal recrudescence ensued. Hamsters that were then given a systemic Mel infusion s.c. immediately initiated a second gonadal regression, implying that neurons at each site become refractory to Mel without compromising responsiveness of other Mel target tissues. Refractoriness occurs locally and independently at each neural target tissue, rather than in a separate “refractoriness” substrate. Restricted, target-specific actions of Mel are consistent with the independent regulation by day length of the several behavioral and physiological traits that vary seasonally in mammals.

Detection of synchrony in the activity of auditory nerve fibers by octopus cells of the mammalian cochlear nucleus

The anatomical and biophysical specializations of octopus cells allow them to detect the coincident firing of groups of auditory nerve fibers and to convey the precise timing of that coincidence to their targets. Octopus cells occupy a sharply defined region of the most caudal and dorsal part of the mammalian ventral cochlear nucleus. The dendrites of octopus cells cross the bundle of auditory nerve fibers just proximal to where the fibers leave the ventral and enter the dorsal cochlear nucleus, each octopus cell spanning about one-third of the tonotopic array. Octopus cells are excited by auditory nerve fibers through the activation of rapid, calcium-permeable, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors. Synaptic responses are shaped by the unusual biophysical characteristics of octopus cells. Octopus cells have very low input resistances (about 7 MΩ), and short time constants (about 200 μsec) as a consequence of the activation at rest of a hyperpolarization-activated mixed-cation conductance and a low-threshold, depolarization-activated potassium conductance. The low input resistance causes rapid synaptic currents to generate rapid and small synaptic potentials. Summation of small synaptic potentials from many fibers is required to bring an octopus cell to threshold. Not only does the low input resistance make individual excitatory postsynaptic potentials brief so that they must be generated within 1 msec to sum but also the voltage-sensitive conductances of octopus cells prevent firing if the activation of auditory nerve inputs is not sufficiently synchronous and depolarization is not sufficiently rapid. In vivo in cats, octopus cells can fire rapidly and respond with exceptionally well-timed action potentials to periodic, broadband sounds such as clicks. Thus both the anatomical specializations and the biophysical specializations make octopus cells detectors of the coincident firing of their auditory nerve fiber inputs.

Linear and nonlinear pathways of spectral information transmission in the cochlear nucleus

At the level of the cochlear nucleus (CN), the auditory pathway divides into several parallel circuits, each of which provides a different representation of the acoustic signal. Here, the representation of the power spectrum of an acoustic signal is analyzed for two CN principal cells—chopper neurons of the ventral CN and type IV neurons of the dorsal CN. The analysis is based on a weighting function model that relates the discharge rate of a neuron to first- and second-order transformations of the power spectrum. In chopper neurons, the transformation of spectral level into rate is a linear (i.e., first-order) or nearly linear function. This transformation is a predominantly excitatory process involving multiple frequency components, centered in a narrow frequency range about best frequency, that usually are processed independently of each other. In contrast, type IV neurons encode spectral information linearly only near threshold. At higher stimulus levels, these neurons are strongly inhibited by spectral notches, a behavior that cannot be explained by level transformations of first- or second-order. Type IV weighting functions reveal complex excitatory and inhibitory interactions that involve frequency components spanning a wider range than that seen in choppers. These findings suggest that chopper and type IV neurons form parallel pathways of spectral information transmission that are governed by two different mechanisms. Although choppers use a predominantly linear mechanism to transmit tonotopic representations of spectra, type IV neurons use highly nonlinear processes to signal the presence of wide-band spectral features.