Techniques for RNA in vivo imaging in plants

Since the discovery of small RNAs and RNA silencing, RNA biology has taken a centre stage in cell and developmental biology. Small RNAs, but also mRNAs and other types of cellular and viral RNAs are processed at specific subcellular localizations. To fully understand cellular RNA metabolism and the various processes influenced byit, techniques are required that permit the sequence-specific tracking of RNAs in living cells. A variety of methods for RNA visualization have been developed since the 1990s, but plant cells pose particular challenges and not all approaches are applicable to them. On the other hand, plant RNA metabolism is particularly diverse and RNAs are even transported between cells, so RNA imaging can potentially provide many valuable insights into plant function at the cellular and tissue level. This Short Review briefly introduces the currently available techniques for plant RNA in vivo imaging and discusses their suitability for different biological questions.PostprintPeer reviewe

Creating contacts between replication and movement at plasmodesmata – a role for membrane contact sites in plant virus infections?

Parts of this work were funded by the University of Florida, Institute of Food and Agricultural Sciences, Early Career Seed Grant No. 00127818 to AL and by the UK Biotechnology and Biomedical Sciences Research Council (BBSRC) grant BB/M007200/1 to JT.To infect their hosts and cause disease, plant viruses must replicate within cells and move throughout the plant both locally and systemically. RNA virus replication occurs on the surface of various cellular membranes, whose shape and composition become extensively modified in the process. Membrane contact sites (MCS) can mediate non-vesicular lipid-shuttling between different membranes and viruses co-opt components of these structures to make their membrane environment suitable for replication. Whereas animal viruses exit and enter cells when moving throughout their host, the rigid wall of plant cells obstructs this pathway and plant viruses therefore move between cells symplastically through plasmodesmata (PD). PD are membranous channels connecting nearly all plant cells and are now viewed to constitute a specialized type of endoplasmic reticulum (ER)-plasma membrane (PM) MCS themselves. Thus, both replication and movement of plant viruses rely on MCS. However, recent work also suggests that for some viruses, replication and movement are closely coupled at ER-PM MCS at the entrances of PD. Movement-coupled replication at PD may be distinct from the main bulk of replication and virus accumulation, which produces progeny virions for plant-to-plant transmission. Thus, MCS play a central role in plant virus infections, and may provide a link between two essential steps in the viral life cycle, replication and movement. Here, we provide an overview of plant virus-MCS interactions identified to date, and place these in the context of the connection between viral replication and cell-to-cell movement.Publisher PDFPeer reviewe

Why a long-lived fireball can be compatible with HBT measurements

The common interpretation of HBT data measured at top SPS energies leads to apparent source lifetimes of 6-8 fm/c and emission duration of approximately 2-3 fm/c. We investigate a scenario with continuous pion emission from a long-lived (~17 fm/c) thermalized source in order to show that it is not excluded by the data. Starting from a description of the source's spacetime expansion based on gross thermodynamical properties of hot matter (which is able to describe a number of experimental observables), we introduce the pion emission function with a contribution from continuous emission during the source's lifetime and another contribution from the final breakup and proceed by calculating the HBT parameters R_out and R_side. The results are compared with experimental data measured at SPS for 158 AGeV central Pb-Pb collisions. We achieve agreement with data, provided that some minor modifications of the fireball evolution scenario are made and find that the parameter R_out is not sensitive to the fireball lifetime, but only to the duration of the final breakup, in spite of the fact that emission takes place throughout the whole lifetime. We explicitly demonstrate that those findings do not alter previous results obtained within this model.Comment: 13 pages, 5 figures, submitted to Phys. Rev. C. (revised description of fireball expansion

Reticulons 3 and 6 interact with viral movement proteins

Funding; This research was funded by the Science and Technology Facilities Council Programme (grant no. 14230008), a British Biotechnology and Biological Sciences Research Council (grant no. BB/J004987/1 to Professor Chris Hawes), and a Vice-Chancellors Research Fellowship to V.K. Parts of this work were funded by the U.K. Biotechnology and Biomedical Sciences Research Council (BBSRC) grant BB/M007200/1 to J.T. Work in J.T.'s laboratory is supported by the Scottish Government's Rural and Environment Science and Analytical Services Division (RESAS).Plant reticulon (RTN) proteins are capable of constricting membranes and are vital for creating and maintaining tubules in the endoplasmic reticulum (ER), making them prime candidates for the formation of the desmotubule in plasmodesmata (PD). RTN3 and RTN6 have previously been detected in an Arabidopsis PD proteome and have been shown to be present in primary PD at cytokinesis. It has been suggested that RTN proteins form protein complexes with proteins in the PD plasma membrane and desmotubule to stabilize the desmotubule constriction and regulate PD aperture. Viral movement proteins (vMPs) enable the transport of viruses through PD and can be ER-integral membrane proteins or interact with the ER. Some vMPs can themselves constrict ER membranes or localize to RTN-containing tubules; RTN proteins and vMPs could be functionally linked or potentially interact. Here we show that different vMPs are capable of interacting with RTN3 and RTN6 in a membrane yeast two-hybrid assay, coimmunoprecipitation, and Förster resonance energy transfer measured by donor excited-state fluorescence lifetime imaging microscopy. Furthermore, coexpression of the vMP CMV-3a and RTN3 results in either the vMP or the RTN changing subcellular localization and reduces the ability of CMV-3a to open PD, further indicating interactions between the two proteins.Publisher PDFPeer reviewe

Parallele Erlebnisse eines k.u.k. Offiziers an der Ostfront und in russischer Kriegsgefangenschaft sowie seiner Frau in der Heimat im Ersten Weltkrieg

Ausgangspunkt für die vorliegende Arbeit sind die Tagebücher eines k.u.k. Offiziers, der im März 1915 in russische Kriegsgefangenschaft geriet und erst im Dezember 1920 nach Österreich zurückkehrte. Dabei berichtet der Offizier zunächst über den Kriegsalltag in der eingeschlossenen Festung Przemyśl und nach deren Fall über das Leben in sibirischen Kriegsgefangenenlagern. Er erlebte das russische Zarenreich während des Ersten Weltkriegs, das eine noch nie dagewesene Anzahl an Kriegsgefangenen zu versorgen hatte und den Bürgerkrieg in Sibirien. Die zweite entscheidende Quelle für diese Diplomarbeit sind die Tagebücher seiner Ehefrau, deren Aufzeichnungen mit der Geburt der ersten Tochter 1900 beginnen und bis ins Jahr 1918 reichen. Diese Bücher geben Hinweise über das Familienleben des Offiziers vor dem Ausbruch des Ersten Weltkriegs sowie über die Sorge einer Frau um ihren Ehemann, der zunächst im Krieg und anschließend tausende Kilometer getrennt von seiner Familie gefangen gehalten wird. Ziel dieser Diplomarbeit ist die deskriptive Darstellung der Erlebnisse eines Offiziers während des Ersten Weltkriegs und in russischer Kriegsgefangenschaft sowie seiner Frau, die in der Heimat vergeblich auf ihren Mann wartete. Das Einsetzen der vorliegenden Berichte in einen historischen Kontext steht dabei im Fokus des Forschungsinteresses. So soll diese Arbeit wissenschaftlich aufgearbeitete Hintergrundinformationen liefern. Dabei stehen folgende Themen im Fokus: der Kampf um die Festung Przemyśl im Ersten Weltkrieg, das Selbstverständnis eines k.u.k. Offiziers, die Kriegsgefangenschaft während und nach dem Ersten Weltkrieg in Russland, die Russische Revolution und ihre Folgen für den weiteren Verlauf des Krieges und die Gefangenen sowie die Rückkehr der Kriegsgefangenen in die Republik Österreich

Two-Particle Correlations at 40, 80, and 158 AGeV Pb-Au Collisions

In this thesis, a centrality dependent study of tow-particle momentum correlations of like-sign pions (HBT interferometry) from Pb-Au collisions at beam energies of 40, 80 and 158 AGeV is presented. The three-dimensional correlation function C_2, depending on the relative momentum components of the particle pair, was parameterized using a three-dimensional Gaussian, modified to consider the final state Coulomb repulsion. The parameterization was done in the Cartesian out-side-long system as proposed by G. Bertsch and S. Pratt. A differential analysis of the HBT radius parameters as a function of the mean transverse pair momentum k_perp allowed to determine transverse geometrical source sizes of about 5.5 fm to 7 fm, increasing with energy and centrality. The mean transverse flow velocity v_perp was found to be of the order 0.5--0.6 c. For the freeze-out time tau_f values between 6 and 8 fm/c were obtained with a duration of particle emission compatible with zero. The beam energy dependence of the HBT radii provide evidence for a universal pion freeze-out condition. The analyzed data have been recorded with the CERES spectrometer at the Super Proton Synchrotron (SPS) accelerator at CERN

Online Pattern Recognition for the ALICE High Level Trigger

The ALICE High Level Trigger has to process data online, in order to select interesting (sub)events, or to compress data efficiently by modeling techniques.Focusing on the main data source, the Time Projection Chamber (TPC), we present two pattern recognition methods under investigation: a sequential approach "cluster finder" and "track follower") and an iterative approach ("track candidate finder" and "cluster deconvoluter"). We show, that the former is suited for pp and low multiplicity PbPb collisions, whereas the latter might be applicable for high multiplicity PbPb collisions, if it turns out, that more than 8000 charged particles would have to be reconstructed inside the TPC. Based on the developed tracking schemes we show, that using modeling techniques a compression factor of around 10 might be achievableComment: Realtime Conference 2003, Montreal, Canada to be published in IEEE Transactions on Nuclear Science (TNS), 6 pages, 8 figure

Reticulons 3 and 6 interact with viral movement proteins

Plant reticulon proteins (RTN) are capable of constricting membranes and vital for creating and maintaining tubules in the endoplasmic reticulum (ER), making them prime candidates for the formation of the desmotubule in plasmodesmata (PD). RTN3 and RTN6 have previously been detected in an Arabidopsis PD proteome and have been shown to be present in primary PD at cytokinesis. It was suggested that RTN proteins form protein complexes with proteins in the PD plasma membrane and desmotubule to stabilize the desmotubule constriction and regulate PD aperture. Viral Movement Proteins (vMPs) enable the transport of viruses through PD and can be ER-integral membrane proteins or interact with the ER. Some vMPs can themselves constrict ER membranes or localise to RTN-containing tubules; RTN proteins and vMPs could be functionally linked or potentially interact. Here we show that different vMPs are capable of interacting with RTN3 and 6 in a membrane yeast-2-hybrid assay, co-immunoprecipitation and Förster resonance energy transfer measured by donor excited-state fluorescence lifetime imaging microscopy (FRET-FLIM). Furthermore, coexpression of the vMP CMV-3a and RTN3 results in either the vMP or the RTN changing subcellular localisation and reduces the ability of CMV-3a to open PD, further indicating interactions between the two proteins

Real Time Global Tests of the ALICE High Level Trigger Data Transport Framework

The High Level Trigger (HLT) system of the ALICE experiment is an online event filter and trigger system designed for input bandwidths of up to 25 GB/s at event rates of up to 1 kHz. The system is designed as a scalable PC cluster, implementing several hundred nodes. The transport of data in the system is handled by an object-oriented data flow framework operating on the basis of the publisher-subscriber principle, being designed fully pipelined with lowest processing overhead and communication latency in the cluster. In this paper, we report the latest measurements where this framework has been operated on five different sites over a global north-south link extending more than 10,000 km, processing a ``real-time'' data flow.Comment: 8 pages 4 figure

The potential consequences for cell Signaling by a class of NOD-Like Receptor proteins (NLRs) bearing an N-terminal signal sequence

The authors gratefully acknowledge the support of the UK Biotechnology and Biological Sciences Research Council (BBSRC) for the work on cellular 2A-like sequences.Publisher PDFPeer reviewe