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

Spectrally red-shifted fluorescent fiducial markers for optimal drift correction in localization microscopy

Precise drift correction is crucial for every single-molecule localization-based microscopy experiment. We evaluate commonly used fiducial markers such as gold nanoparticles, TetraSpeck microspheres, FluoSpheres and nanodiamonds. We introduce spectrally red-shifted fluorescent particles as optimal fiducial markers. Our concept exploits exciting the fluorescent particles at low efficiency far away from their absorption maximum. A fluorescent particle that is covered by a multitude of dyes will nevertheless yield a bright fiducial signal. This represents a simple yet powerful approach to alleviate common problems in drift corrections caused by photobleaching, sudden signal intensity changes or saturation effects of fiducial markers. We adapt our approach for PALM, sptPALM and DNA-PAINT experiments and demonstrate its simple use for varying imaging conditions in different spectral channels

High-resolution study of a star-forming cluster in the Cep-A HW2 region

Due to its relatively small distance (725 pc), the Cepheus A East star-forming region is an ideal laboratory to study massive star formation processes. Based on its morphology, it has been suggested that the flattened molecular gas distribution around the YSO HW2 may be a 350-AU-radius massive protostellar disk. Goal of our work is to ascertain the nature of this structure. We have employed the Plateau de Bure Interferometer to acquire (sub-)arcsecond-resolution imaging of high-density and shock tracers, such as methyl cyanide (CH3CN) and silicon monoxide (SiO), towards the HW2 position. On the 1-arcsecond (about 725 AU) scale, the flattened distribution of molecular gas around HW2 appears to be due to the projected superposition, on the plane of the sky, of at least three protostellar objects, of which at least one is powering a molecular outflow at a small angle with respect to the line of sight. The presence of a protostellar disk around HW2 is not ruled out, but such structure is likely to be detected on a smaller spatial scale, or using different molecular tracers.Comment: 6 pages, 5 figures, accepted for publication in Astronomy & Astrophysic

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

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

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

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

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

RNAi Methodologies for the Functional Study of Signaling Molecules

RNA interference (RNAi) was investigated with the aim of achieving gene silencing with diverse RNAi platforms that include small interfering RNA (siRNA), short hairpin RNA (shRNA) and antisense oligonucleotides (ASO). Different versions of each system were used to silence the expression of specific subunits of the heterotrimeric signal transducing G-proteins, G alpha i2 and G beta 2, in the RAW 264.7 murine macrophage cell line. The specificity of the different RNA interference (RNAi) platforms was assessed by DNA microarray analysis. Reliable RNAi methodologies against the genes of interest were then developed and applied to functional studies of signaling networks. This study demonstrates a successful knockdown of target genes and shows the potential of RNAi for use in functional studies of signaling molecules