162 research outputs found
Super-resolution imaging and estimation of protein copy numbers at single synapses with DNA-PAINT
In the brain, the strength of each individual synapse is defined by the complement of proteins present or the "local proteome." Activity-dependent changes in synaptic strength are the result of changes in this local proteome and posttranslational protein modifications. Although most synaptic proteins have been identified, we still know little about protein copy numbers in individual synapses and variations between synapses. We use DNA-point accumulation for imaging in nanoscale topography as a single-molecule super-resolution imaging technique to visualize and quantify protein copy numbers in single synapses. The imaging technique provides near-molecular spatial resolution, is unaffected by photobleaching, enables imaging of large field of views, and provides quantitative molecular information. We demonstrate these benefits by accessing copy numbers of surface AMPA-type receptors at single synapses of rat hippocampal neurons along dendritic segments
Prevention of neonatal oxygen-induced brain damage by reduction of intrinsic apoptosis
International audienceWithin the last decade, it became clear that oxygen contributes to the pathogenesis of neonatal brain damage, leading to neurocognitive impairment of prematurely born infants in later life. Recently, we have identified a critical role for receptor-mediated neuronal apoptosis in the immature rodent brain. However, the contribution of the intrinsic apoptotic pathway accompanied by activation of caspase-2 under hyperoxic conditions in the neonatal brain still remains elusive. Inhibition of caspases appears a promising strategy for neuroprotection. In order to assess the influence of specific caspases on the developing brain, we applied a recently developed pentapeptide-based group II caspase inhibitor (5-(2,6-difluorophenoxy)-3(R,S)-(2(S)-(2(S)-(3-methoxycarbonyl-2(S)-(3-m ethyl-2(S)-((quinoline-2-carbonyl)-amino)-butyrylamino)propionylamino) 3-methylbutyrylamino) propionylamino)-4-oxo-pentanoic acid methyl ester; TRP601). Here, we report that elevated oxygen (hyperoxia) triggers a marked increase in active caspase-2 expression, resulting in an initiation of the intrinsic apoptotic pathway with upregulation of key proteins, namely, cytochrome c, apoptosis protease-activating factor-1, and the caspase-independent protein apoptosis-inducing factor, whereas BH3-interacting domain death agonist and the anti-apoptotic protein B-cell lymphoma-2 are downregulated. These results coincide with an upregulation of caspase-3 activity and marked neurodegeneration. However, single treatment with TRP601 at the beginning of hyperoxia reversed the detrimental effects in this model. Hyperoxia-mediated neurodegeneration is supported by intrinsic apoptosis, suggesting that the development of highly selective caspase inhibitors will represent a potential useful therapeutic strategy in prematurely born infants. Cell Death and Disease (2012) 3, e250; doi:10.1038/cddis.2011.133; published online 12 January 201
Path Integral Approach to the Scattering Theory of Quantum Transport
The scattering theory of quantum transport relates transport properties of
disordered mesoscopic conductors to their transfer matrix \bbox{T}. We
introduce a novel approach to the statistics of transport quantities which
expresses the probability distribution of \bbox{T} as a path integral. The
path integal is derived for a model of conductors with broken time reversal
invariance in arbitrary dimensions. It is applied to the
Dorokhov-Mello-Pereyra-Kumar (DMPK) equation which describes
quasi-one-dimensional wires. We use the equivalent channel model whose
probability distribution for the eigenvalues of \bbox{TT}^{\dagger} is
equivalent to the DMPK equation independent of the values of the forward
scattering mean free paths. We find that infinitely strong forward scattering
corresponds to diffusion on the coset space of the transfer matrix group. It is
shown that the saddle point of the path integral corresponds to ballistic
conductors with large conductances. We solve the saddle point equation and
recover random matrix theory from the saddle point approximation to the path
integral.Comment: REVTEX, 9 pages, no figure
Fokker-Planck description of the transfer matrix limiting distribution in the scattering approach to quantum transport
The scattering approach to quantum transport through a disordered
quasi-one-dimensional conductor in the insulating regime is discussed in terms
of its transfer matrix \bbox{T}. A model of one-dimensional wires which
are coupled by random hopping matrix elements is compared with the transfer
matrix model of Mello and Tomsovic. We derive and discuss the complete
Fokker-Planck equation which describes the evolution of the probability
distribution of \bbox{TT}^{\dagger} with system length in the insulating
regime. It is demonstrated that the eigenvalues of \ln\bbox{TT}^{\dagger}
have a multivariate Gaussian limiting probability distribution. The parameters
of the distribution are expressed in terms of averages over the stationary
distribution of the eigenvectors of \bbox{TT}^{\dagger}. We compare the
general form of the limiting distribution with results of random matrix theory
and the Dorokhov-Mello-Pereyra-Kumar equation.Comment: 25 pages, revtex, no figure
Correlative Light- and Electron Microscopy with chemical tags
AbstractCorrelative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25nm in the x–y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells
Establishing live-cell single-molecule localization microscopy imaging and single-particle tracking in the archaeon Haloferax volcanii
In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and indispensable tools for the investigation of cellular biology and in vivo biochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology. Here we have developed a reliable protocol for the study of the archaeon Haloferax volcanii. We have generated an autofluorescence-free H. volcanii strain, evaluated several fluorescent proteins for their suitability to serve as single-molecule fluorescence markers and codon-optimized them to work under optimal H. volcanii cultivation conditions. We found that two of them, Dendra2Hfx and PAmCherry1Hfx, provide state-of-the-art single-molecule imaging. Our strategy is quantitative and allows dual-color imaging of two targets in the same field of view as well as DNA co-staining. We present the first single-molecule localization microscopy (SMLM) images of the subcellular organization and dynamics of two crucial intracellular proteins in living H. volcanii cells, FtsZ1, which shows complex structures in the cell division ring, and RNA polymerase, which localizes around the periphery of the cellular DNA. This work should provide incentive to develop SMLM strategies for other archaeal organisms in the near future.</jats:p
Theory of anyon excitons: Relation to excitons of nu=1/3 and nu=2/3 incompressible liquids
Elementary excitations of incompressible quantum liquids (IQL's) are anyons,
i.e., quasiparticles carrying fractional charges and obeying fractional
statistics. To find out how the properties of these quasiparticles manifest
themselves in the optical spectra, we have developed the anyon exciton model
(AEM) and compared the results with the finite-size data for excitons of nu=1/3
and nu=2/3 IQL's. The model considers an exciton as a neutral composite
consisting of three quasielectrons and a single hole. The AEM works well when
the separation between electron and hole confinement planes, h, is larger than
the magnetic length l. In the framework of the AEM an exciton possesses
momentum k and two internal quantum numbers, one of which can be chosen as the
angular momentum, L, of the k=0 state. Existence of the internal degrees of
freedom results in the multiple branch energy spectrum, crater-like electron
density shape and 120 degrees density correlations for k=0 excitons, and the
splitting of the electron shell into bunches for non-zero k excitons. For h
larger than 2l the bottom states obey the superselection rule L=3m (m are
integers starting from 2), all of them are hard core states. For h nearly 2l
there is one-to-one correspondence between the low-energy spectra found for the
AEM and the many- electron exciton spectra of the nu=2/3 IQL, whereas some
states are absent from the many-electron spectra of the nu=1/3 IQL. We argue
that this striking difference in the spectra originates from the different
populational statistics of the quasielectrons of charge conjugate IQL's and
show that the proper account of the statistical requirements eliminates
excessive states from the spectrum. Apparently, this phenomenon is the first
manifestation of the exclusion statistics in the anyon bound states.Comment: 26 pages with 9 figures, typos correcte
Boolean analysis reveals systematic interactions among low-abundance species in the human gut microbiome
The analysis of microbiome compositions in the human gut has gained increasing interest due to the broader availability of data and functional databases and substantial progress in data analysis methods, but also due to the high relevance of the microbiome in human health and disease. While most analyses infer interactions among highly abundant species, the large number of low-abundance species has received less attention. Here we present a novel analysis method based on Boolean operations applied to microbial co-occurrence patterns. We calibrate our approach with simulated data based on a dynamical Boolean network model from which we interpret the statistics of attractor states as a theoretical proxy for microbiome composition. We show that for given fractions of synergistic and competitive interactions in the model our Boolean abundance analysis can reliably detect these interactions. Analyzing a novel data set of 822 microbiome compositions of the human gut, we find a large number of highly significant synergistic interactions among these low-abundance species, forming a connected network, and a few isolated competitive interactions
Three-Dimensional, Tomographic Super-Resolution Fluorescence Imaging of Serially Sectioned Thick Samples
Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 µm×50 µm×2.5 µm. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy
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