Fusion Pores Live on the Edge.

Biological transmission of vesicular content occurs by opening of a fusion pore. Recent experimental observations have illustrated that fusion pores between vesicles that are docked by an extended flat contact zone are located at the edge (vertex) of this zone. We modeled this experimentally observed scenario by coarse-grained molecular simulations and elastic theory. This revealed that fusion pores experience a direct attraction toward the vertex. The size adopted by the resulting vertex pore strongly depends on the apparent contact angle between the adhered vesicles even in the absence of membrane surface tension. Larger contact angles substantially increase the equilibrium size of the vertex pore. Because the cellular membrane fusion machinery actively docks membranes, it facilitates a collective expansion of the contact zone and increases the contact angle. In this way, the fusion machinery can drive expansion of the fusion pore by free energy equivalents of multiple tens of k <sub>B</sub> T from a distance and not only through the fusion proteins that reside within the fusion pore

Non-Hydrolysable Analogues of Cyclic and Branched Condensed Phosphates: Chemistry and Chemical Proteomics.

Studies into the biology of condensed phosphates almost exclusively cover linear polyphosphates. However, there is evidence for the presence of cyclic polyphosphates (metaphosphates) in organisms and for enzymatic digestion of branched phosphates (ultraphosphates) with alkaline phosphatase. Further research of non-linear condensed phosphates in biology would profit from interactome data of such molecules, however, their stability in biological media is limited. Here we present syntheses of modified, non-hydrolysable analogues of cyclic and branched condensed phosphates, called meta- and ultraphosphonates, and their application in a chemical proteomics approach using yeast cell extracts. We identify putative interactors with overlapping hits for structurally related capture compounds underlining the quality of our results. The datasets serve as starting point to study the biological relevance and functions of meta- and ultraphosphates. In addition, we examine the reactivity of meta- and ultraphosphonates with implications for their "hydrolysable" analogues: Efforts to increase the ring-sizes of meta- or cyclic ultraphosphonates revealed a strong preference to form trimetaphosphate-analogue structures by cyclization and/or ring-contraction. Using carbodiimides for condensation, the so far inaccessible dianhydro product of ultraphosphonate, corresponding to P <sub>4</sub> O <sub>11</sub> <sup>2-</sup> , was selectively obtained and then ring-opened by different nucleophiles yielding modified cyclic ultraphosphonates

Cryo-EM structure of the polyphosphate polymerase VTC reveals coupling of polymer synthesis to membrane transit.

The eukaryotic vacuolar transporter chaperone (VTC) complex acts as a polyphosphate (polyP) polymerase that synthesizes polyP from adenosine triphosphate (ATP) and translocates polyP across the vacuolar membrane to maintain an intracellular phosphate (P <sub>i</sub> ) homeostasis. To discover how the VTC complex performs its function, we determined a cryo-electron microscopy structure of an endogenous VTC complex (Vtc4/Vtc3/Vtc1) purified from Saccharomyces cerevisiae at 3.1 Å resolution. The structure reveals a heteropentameric architecture of one Vtc4, one Vtc3, and three Vtc1 subunits. The transmembrane region forms a polyP-selective channel, likely adopting a resting state conformation, in which a latch-like, horizontal helix of Vtc4 limits the entrance. The catalytic Vtc4 central domain is located on top of the pseudo-symmetric polyP channel, creating a strongly electropositive pathway for nascent polyP that can couple synthesis to translocation. The SPX domain of the catalytic Vtc4 subunit positively regulates polyP synthesis by the VTC complex. The noncatalytic Vtc3 regulates VTC through a phosphorylatable loop. Our findings, along with the functional data, allow us to propose a mechanism of polyP channel gating and VTC complex activation

SNARE-mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle.

Constitutive membrane fusion within eukaryotic cells is thought to be controlled at its initial steps, membrane tethering and SNARE complex assembly, and to rapidly proceed from there to full fusion. Although theory predicts that fusion pore expansion faces a major energy barrier and might hence be a rate-limiting and regulated step, corresponding states with non-expanding pores are difficult to assay and have remained elusive. Here, we show that vacuoles in living yeast are connected by a metastable, non-expanding, nanoscopic fusion pore. This is their default state, from which full fusion is regulated. Molecular dynamics simulations suggest that SNAREs and the SM protein-containing HOPS complex stabilize this pore against re-closure. Expansion of the nanoscopic pore to full fusion can thus be triggered by osmotic pressure gradients, providing a simple mechanism to rapidly adapt organelle volume to increases in its content. Metastable, nanoscopic fusion pores are then not only a transient intermediate but can be a long-lived, physiologically relevant and regulated state of SNARE-dependent membrane fusion

2008-2009 President\u27s Report

The Linfield College President\u27s Annual Report is a collection of information about the year in review, including academics, student life and athletics, enrollment, finances, philanthropy, and leadership

First results of the air shower experiment KASCADE

The main goals of the KASCADE (KArlsruhe Shower Core and Array DEtector) experiment are the determination of the energy spectrum and elemental composition of the charged cosmic rays in the energy range around the knee at ca. 5 PeV. Due to the large number of measured observables per single shower a variety of different approaches are applied to the data, preferably on an event-by-event basis. First results are presented and the influence of the high-energy interaction models underlying the analyses is discussed.Comment: 3 pages, 3 figures included, to appear in the TAUP 99 Proceedings, Nucl. Phys. B (Proc. Suppl.), ed. by M. Froissart, J. Dumarchez and D. Vignau

Inositol pyrophosphates activate the vacuolar transport chaperone complex in yeast by disrupting a homotypic SPX domain interaction.

Many proteins involved in eukaryotic phosphate homeostasis are regulated by SPX domains. In yeast, the vacuolar transporter chaperone (VTC) complex contains two such domains, but mechanistic details of its regulation are not well understood. Here, we show at the atomic level how inositol pyrophosphates interact with SPX domains of subunits Vtc2 and Vtc3 to control the activity of the VTC complex. Vtc2 inhibits the catalytically active VTC subunit Vtc4 by homotypic SPX-SPX interactions via the conserved helix α1 and the previously undescribed helix α7. Binding of inositol pyrophosphates to Vtc2 abrogates this interaction, thus activating the VTC complex. Accordingly, VTC activation is also achieved by site-specific point mutations that disrupt the SPX-SPX interface. Structural data suggest that ligand binding induces reorientation of helix α1 and exposes the modifiable helix α7, which might facilitate its post-translational modification in vivo. The variable composition of these regions within the SPX domain family might contribute to the diversified SPX functions in eukaryotic phosphate homeostasis

Electron, Muon, and Hadron Lateral Distributions Measured in Air-Showers by the KASCADE Experiment

Measurements of electron, muon, and hadron lateral distributions of extensive air showers as recorded by the KASCADE experiment are presented. The data cover the energy range from about 5x10^14 eV up to almost 10^17 eV and extend from the inner core region to distances of 200 m. The electron and muon distributions are corrected for mutual contaminations by taking into account the detector properties in the experiment. All distributions are well described by NKG-functions. The scale radii describing the electron and hadron data best are approx. 30 m and 10 m, respectively. We discuss the correlation between scale radii and `age' parameter as well as their dependence on shower size, zenith angle, and particle energy threshold.Comment: 28 pages, 14 figures, Accepted for publication in Astroparticle Physic

KASCADE: Astrophysical results and tests of hadronic interaction models

KASCADE is a multi-detector setup to get redundant information on single air shower basis. The information is used to perform multiparameter analyses to solve the threefold problem of the reconstruction of (i)the unknown primary energy, (ii) the primary mass, and (iii) to quantify the characteristics of the hadronic interactions in the air-shower development. In this talk recent results of the KASCADE data analyses are summarized concerning cosmic ray anisotropy studies, determination of flux spectra for different primary mass groups, and approaches to test hadronic interaction models. Neither large scale anisotropies nor point sources were found in the KASCADE data set. The energy spectra of the light element groups result in a knee-like bending and a steepening above the knee. The topology of the individual knee positions shows a dependency on the primary particle. Though no hadronic interaction model is fully able to describe the multi-parameter data of KASCADE consistently, the more recent models or improved versions of older models reproduce the data better than few years ago.Comment: to appear in Nucl. Phys. B (Proc. Suppl.), Proc. of the XIII ISVHECRI, Pylos 2004 - with a better quality of the figure