Chromatin modification by DNA tracking

In general, the transcriptional competence of a chromatin domain is correlated with increased sensitivity to DNase I cleavage. A recent observation that actively transcribing RNA polymerase II piggybacks a histone acetyltranferase activity [Wittschieben, B., Otero, G., de Bizemont, T., Fellows, J., Erdjument-Bromage, H., Ohba, R., Li, Y., Allis, C. D., Tempst, P. & Svejstrup, J. Q. (1999) Mol. Cell 4, 123–128] implies that the state of histone acetylation, and hence the ability of chromatin to fold, can be altered by a processive mechanism. In this article, it is proposed that tracking-mediated chromatin modification could create and/or maintain an open configuration in a complete chromatin domain including both intra- and extragenic regions. This mechanism suggests a putative functional role for the extragenic transcription observed at the β-globin and other loci in vertebrate cells.

Tracking molecular evolution of photosynthesis by characterization of a major photosynthesis gene cluster from Heliobacillus mobilis

A DNA sequence has been obtained for a 35.6-kb genomic segment from Heliobacillus mobilis that contains a major cluster of photosynthesis genes. A total of 30 ORFs were identified, 20 of which encode enzymes for bacteriochlorophyll and carotenoid biosynthesis, reaction-center (RC) apoprotein, and cytochromes for cyclic electron transport. Donor side electron-transfer components to the RC include a putative RC-associated cytochrome c553 and a unique four-large-subunit cytochrome bc complex consisting of Rieske Fe-S protein (encoded by petC), cytochrome b6 (petB), subunit IV (petD), and a diheme cytochrome c (petX). Phylogenetic analysis of various photosynthesis gene products indicates a consistent grouping of oxygenic lineages that are distinct and descendent from anoxygenic lineages. In addition, H. mobilis was placed as the closest relative to cyanobacteria, which form a monophyletic origin to chloroplast-based photosynthetic lineages. The consensus of the photosynthesis gene trees also indicates that purple bacteria are the earliest emerging photosynthetic lineage. Our analysis also indicates that an ancient gene-duplication event giving rise to the paralogous bchI and bchD genes predates the divergence of all photosynthetic groups. In addition, our analysis of gene duplication of the photosystem I and photosystem II core polypeptides supports a “heterologous fusion model” for the origin and evolution of oxygenic photosynthesis.

Neurotransplantation of magnetically labeled oligodendrocyte progenitors: Magnetic resonance tracking of cell migration and myelination

Demyelination is a common pathological finding in human neurological diseases and frequently persists as a result of failure of endogenous repair. Transplanted oligodendrocytes and their precursor cells can (re)myelinate axons, raising the possibility of therapeutic intervention. The migratory capacity of transplanted cells is of key importance in determining the extent of (re)myelination and can, at present, be evaluated only by using invasive and irreversible procedures. We have exploited the transferrin receptor as an efficient intracellular delivery device for magnetic nanoparticles, and transplanted tagged oligodendrocyte progenitor cells into the spinal cord of myelin-deficient rats. Cell migration could be easily detected by using three-dimensional magnetic resonance microscopy, with a close correlation between the areas of contrast enhancement and the achieved extent of myelination. The present results demonstrate that magnetic resonance tracking of transplanted oligodendrocyte progenitors is feasible; this technique has the potential to be easily extended to other neurotransplantation studies involving different precursor cell types.

Fluorescent RNA cytochemistry: tracking gene transcripts in living cells

The advent of jellyfish green fluorescent protein and its spectral variants, together with promising new fluorescent proteins from other classes of the Cnidarian phylum (coral and anemones), has greatly enhanced and promises to further boost the detection and localization of proteins in cell biology. It has been less widely appreciated that highly sensitive methods have also recently been developed for detecting the movement and localization in living cells of the very molecules that precede proteins in the gene expression pathway, i.e. RNAs. These approaches include the microinjection of fluorescent RNAs into living cells, the in vivo hybridization of fluorescent oligonucleotides to endogenous RNAs and the expression in cells of fluorescent RNA-binding proteins. This new field of ‘fluorescent RNA cytochemistry’ is summarized in this article, with emphasis on the biological insights it has already provided. These new techniques are likely to soon collaborate with other emerging approaches to advance the investigation of RNA birth, RNA–protein assembly and ribonucleoprotein particle transport in systems such as oocytes, embryos, neurons and other somatic cells, and may even permit the observation of viral replication and transcription pathways as they proceed in living cells, ushering in a new era of nucleic acids research in vivo.

Tracking memory's trace

There is strong converging evidence that the intermediate and medial part of the hyperstriatum ventrale of the chick brain is a memory store for information acquired through the learning process of imprinting. Neurons in this memory system come, through imprinting, to respond selectively to the imprinting stimulus (IS) neurons and so possess the properties of a memory trace. Therefore, the responses of the intermediate and medial part of the hyperstriatum ventrale neurons to a visual imprinting stimulus were determined before, during, and after training. Of the total recorded population, the proportions of IS neurons shortly after each of two 1-h training sessions were significantly higher (approximately 2 times) than the pretraining proportion. However, ≈4.5 h later this proportion had fallen significantly and did not differ significantly from the pretraining proportion. Nevertheless, ≈21.5 h after the end of training, the proportion of IS neurons was at its highest (approximately 3 times the pretraining level). No significant fluctuations occurred in the proportions of neurons responding to the alternative stimulus. In addition, nonmonotonic changes were found commonly in the activity of 230 of the neurons tracked individually from before training to shortly after the end of training. Thus the pattern of change in responsiveness both at the population level and at the level of individual neurons was highly nonmonotonic. Such a pattern of change is not consistent with simple models of memory based on synaptic strengthening to asymptote. A model is proposed that accounts for the changes in the population responses to the imprinting stimulus in terms of changes in the responses of individual neurons.

Tracking the estrogen receptor in neurons: Implications for estrogen-induced synapse formation

Estrogens (E) and progestins regulate synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat, and the functional consequences include changes in neurotransmission and memory. Synapse formation has been demonstrated by using the Golgi technique, dye filling of cells, electron microscopy, and radioimmunocytochemistry. N-methyl-d-aspartate (NMDA) receptor activation is required, and inhibitory interneurons play a pivotal role as they express nuclear estrogen receptor alpha (ERα) and show E-induced decreases of GABAergic activity. Although global decreases in inhibitory tone may be important, a more local role for E in CA1 neurons seems likely. The rat hippocampus expresses both ERα and ERβ mRNA. At the light microscopic level, autoradiography shows cell nuclear [3H]estrogen and [125I]estrogen uptake according to a distribution that primarily reflects the localization of ERα-immunoreactive interneurons in the hippocampus. However, recent ultrastructural studies have revealed extranuclear ERα immunoreactivity (IR) within select dendritic spines on hippocampal principal cells, axon terminals, and glial processes, localizations that would not be detectable by using standard light microscopic methods. Based on recent studies showing that both types of ER are expressed in a form that activates second messenger systems, these findings support a testable model in which local, non-genomic regulation by estrogen participates along with genomic actions of estrogens in the regulation of synapse formation.

Tracking wakes: The nocturnal predatory strategy of piscivorous catfish

Swimming fish leave wakes containing hydrodynamic and chemical traces. These traces mark their swim paths and could guide predators. We now show that nocturnal European catfish (Silurus glanis) locate a piscine prey (guppy, Poecilia reticulata) by accurately tracking its three-dimensional swim path before an attack in the absence of visible light. Wakes that were up to 10 s old were followed over distances up to 55 prey-body lengths in our setup. These results demonstrate that prey wakes remain sufficiently identifiable to guide predators, and to extend considerably the area in which prey is detectable. Moreover, wakes elicit rear attacks, which may be more difficult to detect by prey. Wake tracking may be a common strategy among aquatic predators.

In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function.

To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.

Three-dimensional tracking of motile bacteria near a solid planar surface.

Knowing how motile bacteria move near and along a solid surface is crucial to understanding such diverse phenomena as the migration of infectious bacteria along a catheter, biofilm growth, and the movement of bacteria through the pore spaces of saturated soil, a critical step in the in situ bioremediation of contaminated aquifers. In this study, a tracking microscope is used to record the three-dimensional motion of Escherichia coli near a planar glass surface. Data from the tracking microscope are analyzed to quantify the effects of bacteria-surface interactions on the swimming behavior of bacteria. The speed of cells approaching the surface is found to decrease in agreement with the mathematical model of Ramia et al. [Ramia, M., Tullock, D. L. & Phan-Tien, N. (1993) Biophys J. 65,755-778], which represents the bacteria as spheres with a single polar flagellum rotating at a constant rate. The tendency of cells to swim adjacent to the surface is shown in computer-generated reproductions of cell traces. The attractive interaction potential between the cells and the solid surface is offered as one of several possible explanations for this tendency.