Tracking single molecules in the live cell plasma membrane-Do's and Don't's

In recent years, the development of fast and highly sensitive microscopy has changed the way of thinking of cell biologists: it became more and more important to study the structural origin for cellular function, and industry turned its attention to the improvement of the required instruments. Optical microscopy has now reached a milestone in sensitivity by resolving the signal of a single, fluorescence-labeled biomolecule within a living cell. First steps towards these pioneering studies were set by methods developed in the late eighties for tracking single biomolecules labeled with fluorescent latex spheres or gold-particles. Meanwhile, a time-resolution of milliseconds for imaging weakly fluorescent cellular structures like small organelles, vesicles, or even single molecules is state-of-the-art. The advances in the fields of microscopy brought new cell biological questions into reach. The investigation of a single fluorescent molecule-or simultaneously of an ensemble of individual molecules-provides principally new information, which is generally hidden in ensemble-averaged signals of molecules. In this paper we describe strategies how to make use of single molecule trajectories for deducing information about nanoscopic structures in a live cell context. In particular, we focus our discussion on elucidating the plasma membrane organization by single molecule tracking. A diffusing membrane constituent--e.g. a protein or a lipid--experiences a manifold of interactions on its path: the most rapid interactions represent the driving force for free diffusion; stronger or correlated interactions can be frequently observed as subdiffusive behavior. Correct interpretation of the data has the potential to shine light on this enigmatic organelle, where membrane rafts, protein microdomains, fences and pickets still frolic through the text-book sketches. We summarize available analytical models and point out potential pitfalls, which may result in quantitative or three even qualitative misinterpretations.     

The predictive power of the CluSTr database

SUMMARY: The CluSTr database employs a fully automatic single-linkage hierarchical clustering method based on a similarity matrix. In order to compute the matrix, first all-against-all pair-wise comparisons between protein sequences are computed using the Smith-Waterman algorithm. The statistical significance of the similarity scores is then assessed using a Monte Carlo analysis, yielding Z-values, which are used to populate the matrix. This paper describes automated annotation experiments that quantify the predictive power and hence the biological relevance of the CluSTr data. The experiments utilized the UniProt data-mining framework to derive annotation predictions using combinations of InterPro and CluSTr. We show that this combination of data sources greatly increases the precision of predictions made by the data-mining framework, compared with the use of InterPro data alone. We conclude that the CluSTr approach to clustering proteins makes a valuable contribution to traditional protein classifications. AVAILABILITY: http://www.ebi.ac.uk/clustr/.     

The number of functional olfactory receptor genes and the relative size of olfactory brain structures are poor predictors of olfactory discrimination performance with enantiomers

The ability of four squirrel monkeys and three pigtail macaques to distinguish between nine enantiomeric odor pairs sharing an isopropenyl group at the chiral center was investigated in terms of a conditioning paradigm. All animals from both species were able to discriminate between the optical isomers of limonene, carvone, dihydrocarvone, dihydrocarveole and dihydrocarvyl acetate, whereas they failed to distinguish between the (+)- and (-)-forms of perillaaldehyde and limonene oxide. The pigtail macaques, but not the squirrel monkeys, also discriminated between the antipodes of perillaalcohol and isopulegol. A comparison of the across-task patterns of discrimination performance shows a high degree of similarity among the two primate species and also between these nonhuman primates and human subjects tested in an earlier study on the same tasks. These findings suggest that between-species comparisons of the relative size of olfactory brain structures or of the number of functional olfactory receptor genes are poor predictors of olfactory discrimination performance with enantiomers.     

Olfactory responsiveness to two odorous steroids in three species of nonhuman primates

Social communication by means of odor signals is widespread among mammals. In pigs, for example, the C19-steroids 5-alpha-androst-16-en-3-one and 5-alpha-androst-16-en-3-ol are secreted by the boar and induce the mating stance in the sow. In humans, the same substances have been shown to be compounds of body odor and are presumed to affect human behavior. Using an instrumental conditioning paradigm, we here show that squirrel monkeys, spider monkeys and pigtail macaques are able to detect androstenone at concentrations in the micromolar range and thus at concentrations at least as low as those reported in pigs and humans. All three species of nonhuman primates were considerably less sensitive to androstenol, which was detected at concentrations in the millimolar range. Additional tests, using a habituation-dishabituation paradigm, showed that none of the 10 animals tested per species was anosmic to the two odorous steroids. These results suggest that androstenone and androstenol may be involved in olfactory communication in the primate species tested and that the specific anosmia to these odorants found in approximately 30% of human subjects may be due to their reduced number of functional olfactory receptor genes compared with nonhuman primates.     

Confirmation of the type 2 myotonic dystrophy (CCTG)n expansion mutation in patients with proximal myotonic myopathy/proximal myotonic dystrophy of different European origins: a single shared haplotype indicates an ancestral founder effect

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, is a clinically and genetically heterogeneous neuromuscular disorder. DM is characterized by autosomal dominant inheritance, muscular dystrophy, myotonia, and multisystem involvement. Type 1 DM (DM1) is caused by a (CTG)(n) expansion in the 3' untranslated region of DMPK in 19q13.3. Multiple families, predominantly of German descent and with clinically variable presentation that included proximal myotonic myopathy (PROMM) and type 2 DM (DM2) but without the DM1 mutation, showed linkage to the 3q21 region and were recently shown to segregate a (CCTG)(n) expansion mutation in intron 1 of ZNF9. Here, we present linkage to 3q21 and mutational confirmation in 17 kindreds of European origin with PROMM and proximal myotonic dystrophy, from geographically distinct populations. All patients have the DM2 (CCTG)(n) expansion. To study the evolution of this mutation, we constructed a comprehensive physical map of the DM2 region around ZNF9. High-resolution haplotype analysis of disease chromosomes with five microsatellite and 22 single-nucleotide polymorphism markers around the DM2 mutation identified extensive linkage disequilibrium and a single shared haplotype of at least 132 kb among patients from the different populations. With the exception of the (CCTG)(n) expansion, the available markers indicate that the DM2 haplotype is identical to the most common haplotype in normal individuals. This situation is reminiscent of that seen in DM1. Taken together, these data suggest a single founding mutation in DM2 patients of European origin. We estimate the age of the founding haplotype and of the DM2 (CCTG) expansion mutation to be approximately 200-540 generations.     

DNA interaction with Mn2+ ions at elevated temperatures: VCD evidence of DNA aggregation

Interaction of natural calf thymus DNA with Mn(2+) ions was studied at room temperature and at elevated temperatures in the range from 23 degrees C to 94 degrees C by means of IR absorption and vibrational circular dichroism (VCD) spectroscopy. The Mn(2+) concentration was varied between 0 and 1.3M (0 and 10 [Mn]/[P]). The secondary structure of DNA remained in the frame of the B-form family in the whole ion concentration range at room temperature. No significant DNA denaturation was revealed at room temperature even at the highest concentration of metal ions studied. However at elevated temperatures, DNA denaturation and a significant decrease of the melting temperature of DNA connected with a decrease of the stability of DNA induced by Mn(2+) ions occurred. VCD demonstrated sensitivity to DNA condensation and aggregation as well as an ability to distinguish between these two processes. No condensation or aggregation of DNA was observed at room temperature at any of the metal ion concentrations studied. DNA condensation was revealed in a very narrow range of experimental conditions at around 2.4 [Mn]/[P] and about 55 degrees C. DNA aggregation was observed in the presence of Mn(2+) ions at elevated temperatures during or after denaturation. VCD spectroscopy turned out to be useful for studying DNA condensation and aggregation due to its ability to distinguish between these two processes, and for providing information about DNA secondary structure in a condensed or aggregated state.     

Cisplatin adducts of d(CCTCTG*G*TCTCC).d(GGAGACCAGAGG) in aqueous solution by vibrational circular dichroism spectroscopy

The vibrational circular dichroism (VCD) and IR absorption spectra of a dodecamer d(CCTCTGGTCTCC).d(GGAGACCAGAGG) coordinated with cisplatin are distinct compared to those of the dodecamer without cisplatin. Although the intensity of PO(2)/deoxyribose absorptions (1150-850 cm(-1)) increases noticeably relative to those of the carbonyl and ring deformations of the bases (1750-1500 cm(-1)), the VCD spectra differ to a much greater extent. Overlapping positive and negative bands can be assigned relatively easily to individual vibrational modes. The effect of platination on the dodecamer duplex is expressed most prominently in VCD arising solely from the vibrations of the guanines bound to the platinum atom. The effect on the VCD features of other bases leads to minute wavenumber shifts at most. These observations are in agreement with previous NMR and X-ray experiments on the same oligonucleotide. The assignment of the absorption and VCD bands strongly resembles those of the octamer duplex d(CCTGGTCC).d(GGACCAGG) when coordinated with platinum. The spectra of the dodecamer did not indicate any isomerization of the complex with time, as is clearly the case for the octamer.     

Growth and metabolism in a marine nematode,Enoplus communis Bastian

Summary The life history and metabolism of a marine nematode, Enoplus communis Bastian, was studied. The species has an annual life cycle, spawning taking place in early spring. Maturity is reached in fall and early winter, and both sexes overwinter with their gonads fully developed.The metabolism is not temperature-compensated and there is no difference in respiration between the two sexes.The respiration rate is fairly low in the fertilized, uncleaved eggs (about 600 mm³ O₂/g/hr). Right after hatching, the oxygen consumption increases sharply to a value about three times that of the eggs. Then it drops quickly, but in animals weighing between 6 and 10 g, another sudden rise occurs, followed by a second drop and subsequent leveling of the curve. The first increase in metabolic rate from egg to first juvenile stage might be due to the activation of metabolically important substances. The following changes, however, are most likely correlated with the process of molting and are probably due to changes in the percentage of dry tissue matter in animals prior to and after ecdysis.The large fluctuations in respiration rate occuring in animals of different size, despite a constant surface/volume ratio, suggest that body surface proper does not control oxygen consumption in this species.Contribution no. 1059 from the Woods Hole Oceanographic Institution, Woods Hole, Mass.Supported by NSF grant G-4813.     

Free Radical Scavengers and Photosynthetic Pigments in Pinus Cembra L. Needles as Affected by Ozone Exposure

The goal of this study was the characterization of the antioxidative protection system of current and 1-year-old needles of a cembran pine (Pinus cembra L.) and its possible responses to elevated concentrations of atmospheric O₃. Twigs of a mature cembran pine at the alpine timberline (1950 m a.s.l.) were exposed in climate-controlled twig chambers for 91 d to charcoal-filtered air (CF), ambient air O₃ concentration (A), and two-fold ambient air O₃ concentration (2A). Additionally, a chamberless control group (AA) was used to examine chamber effects. At the end of the fumigation period the contents of free radical scavengers and photosynthetic pigments were measured in the needles. Independent from O₃ exposure, total ascorbate and -tocopherol contents were higher in 1-year-old needles compared to the current flush while the opposite was found for glutathione. The amounts of pigments and antioxidants in P. cembra needles were comparable to those in other conifers growing at high-elevation sites. The only hint toward O₃ induced changes in the composition of antioxidants was an increase in the glutathione redox state toward more oxidation in 1-year-old needles upon exposure to A or AA conditions, but not upon 2A exposure. Chlorophyll and carotenoid contents were not affected by O₃ neither in current- nor in previous-year needles. The de-epoxidation state of the xanthophyll cycle pigments, however, was significantly increased in 1-year-old needles under A and AA compared to the CF control, but not under 2A. Hence, Pinus cembra, which is well adapted to the extreme environment of the timberline ecotone, exhibited only marginal biochemical changes in response to elevated O₃.     

Vibrational CD (VCD) and atomic force microscopy (AFM) study of DNA interaction with Cr3+ ions: VCD and AFM evidence of DNA condensation

The interaction of natural calf thymus DNA with Cr(3+) ions was studied at room temperature by means of vibrational CD (VCD) and infrared absorption (ir) spectroscopy, and atomic force microscopy (AFM). Cr(3+) ion binding mainly to N(7) (G) and to phosphate groups was demonstrated. Psi-type VCD spectra resembling electronic CD (ECD) spectra, which appear during psi-type DNA condensation, were observed. These spectra are characterized mainly by an anomalous, severalfold increase of VCD intensity. Such anomalous VCD spectra were assigned to DNA condensation with formation of large and dense particles of a size comparable to the wavelength of the probing ir beam and possessing large-scale helicity. Atomic force microscopy confirmed DNA condensation by Cr(3+) ions and the formation of tight DNA particles responsible for the psi-type VCD spectra. Upon increasing the Cr(3+) ion concentration the shape of the condensates changed from loose flower-like structures to highly packed dense spheres. No DNA denaturation was seen even at the highest concentration of Cr(3+) ions studied. The secondary structure of DNA remained in a B-form before and after the condensation. VCD and ir as well as AFM proved to be an effective combination for investigating DNA condensation. In addition to the ability of VCD to determine DNA condensation, VCD and ir can in the same experiment provide unambiguous information about the secondary structure of DNA contained in the condensed particles.