High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the Yeast Saccharomyces cerevisiaeV⃞

The spindle pole body (SPB) is the major microtubule-organizing center of budding yeast and is the functional equivalent of the centrosome in higher eukaryotic cells. We used fast-frozen, freeze-substituted cells in conjunction with high-voltage electron tomography to study the fine structure of the SPB and the events of early spindle formation. Individual structures were imaged at 5–10 nm resolution in three dimensions, significantly better than can be achieved by serial section electron microscopy. The SPB is organized in distinct but coupled layers, two of which show ordered two-dimensional packing. The SPB central plaque is anchored in the nuclear envelope with hook-like structures. The minus ends of nuclear microtubules (MTs) are capped and are tethered to the SPB inner plaque, whereas the majority of MT plus ends show a distinct flaring. Unbudded cells containing a single SPB retain 16 MTs, enough to attach to each of the expected 16 chromosomes. Their median length is ∼150 nm. MTs growing from duplicated but not separated SPBs have a median length of ∼130 nm and interdigitate over the bridge that connects the SPBs. As a bipolar spindle is formed, the median MT length increases to ∼300 nm and then decreases to ∼30 nm in late anaphase. Three-dimensional models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complement and extend what is known about the three-dimensional structure of the yeast mitotic spindle and further our understanding of the organization of the SPB in intact cells.

Secretagogue-triggered Transfer of Membrane Proteins from Neuroendocrine Secretory Granules to Synaptic-like Microvesicles

The membrane proteins of all regulated secretory organelles (RSOs) recycle after exocytosis. However, the recycling of those membrane proteins that are targeted to both dense core granules (DCGs) and synaptic-like microvesicles (SLMVs) has not been addressed. Since neuroendocrine cells contain both RSOs, and the recycling routes that lead to either organelle overlap, transfer between the two pools of membrane proteins could occur during recycling. We have previously demonstrated that a chimeric protein containing the cytosolic and transmembrane domains of P-selectin coupled to horseradish peroxidase is targeted to both the DCG and the SLMV in PC12 cells. Using this chimera, we have characterized secretagogue-induced traffic in PC12 cells. After stimulation, this chimeric protein traffics from DCGs to the cell surface, internalizes into transferrin receptor (TFnR)-positive endosomes and thence to a population of secretagogue-responsive SLMVs. We therefore find a secretagogue-dependent rise in levels of HRP within SLMVs. In addition, the levels within SLMVs of the endogenous membrane protein, synaptotagmin, as well as a green fluorescent protein-tagged version of vesicle-associated membrane protein (VAMP)/synaptobrevin, also show a secretagogue-dependent increase.

Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

Topogenic determinants that direct protein topology at the endoplasmic reticulum membrane usually function with high fidelity to establish a uniform topological orientation for any given polypeptide. Here we show, however, that through the coupling of sequential translocation events, native topogenic determinants are capable of generating two alternate transmembrane structures at the endoplasmic reticulum membrane. Using defined chimeric and epitope-tagged full-length proteins, we found that topogenic activities of two C-trans (type II) signal anchor sequences, encoded within the seventh and eighth transmembrane (TM) segments of human P-glycoprotein were directly coupled by an inefficient stop transfer (ST) sequence (TM7b) contained within the C-terminus half of TM7. Remarkably, these activities enabled TM7 to achieve both a single- and a double-spanning TM topology with nearly equal efficiency. In addition, ST and C-trans signal anchor activities encoded by TM8 were tightly linked to the weak ST activity, and hence topological fate, of TM7b. This interaction enabled TM8 to span the membrane in either a type I or a type II orientation. Pleiotropic structural features contributing to this unusual topogenic behavior included 1) a short, flexible peptide loop connecting TM7a and TM7b, 2) hydrophobic residues within TM7b, and 3) hydrophilic residues between TM7b and TM8.

Uracil-DNA glycosylase–DNA substrate and product structures: Conformational strain promotes catalytic efficiency by coupled stereoelectronic effects

Enzymatic transformations of macromolecular substrates such as DNA repair enzyme/DNA transformations are commonly interpreted primarily by active-site functional-group chemistry that ignores their extensive interfaces. Yet human uracil–DNA glycosylase (UDG), an archetypical enzyme that initiates DNA base-excision repair, efficiently excises the damaged base uracil resulting from cytosine deamination even when active-site functional groups are deleted by mutagenesis. The 1.8-Å resolution substrate analogue and 2.0-Å resolution cleaved product cocrystal structures of UDG bound to double-stranded DNA suggest enzyme–DNA substrate-binding energy from the macromolecular interface is funneled into catalytic power at the active site. The architecturally stabilized closing of UDG enforces distortions of the uracil and deoxyribose in the flipped-out nucleotide substrate that are relieved by glycosylic bond cleavage in the product complex. This experimentally defined substrate stereochemistry implies the enzyme alters the orientation of three orthogonal electron orbitals to favor electron transpositions for glycosylic bond cleavage. By revealing the coupling of this anomeric effect to a delocalization of the glycosylic bond electrons into the uracil aromatic system, this structurally implicated mechanism resolves apparent paradoxes concerning the transpositions of electrons among orthogonal orbitals and the retention of catalytic efficiency despite mutational removal of active-site functional groups. These UDG/DNA structures and their implied dissociative excision chemistry suggest biology favors a chemistry for base-excision repair initiation that optimizes pathway coordination by product binding to avoid the release of cytotoxic and mutagenic intermediates. Similar excision chemistry may apply to other biological reaction pathways requiring the coordination of complex multistep chemical transformations.

Stability of nonrelativistic quantum mechanical matter coupled to the (ultraviolet cutoff) radiation field

We announce a proof of H-stability for the quantized radiation field, with ultraviolet cutoff, coupled to arbitrarily many non-relativistic quantized electrons and static nuclei. Our result holds for arbitrary atomic numbers and fine structure constant. We also announce bounds for the energy of many electrons and nuclei in a classical vector potential and for the eigenvalue sum of a one-electron Pauli Hamiltonian with magnetic field.

Brittle-1, an Adenylate Translocator, Facilitates Transfer of Extraplastidial Synthesized ADP-Glucose into Amyloplasts of Maize Endosperms1

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.

The Regulation of Photosynthetic Electron Transport during Nutrient Deprivation in Chlamydomonas reinhardtii1

The light-saturated rate of photosynthetic O2 evolution in Chlamydomonas reinhardtii declined by approximately 75% on a per-cell basis after 4 d of P starvation or 1 d of S starvation. Quantitation of the partial reactions of photosynthetic electron transport demonstrated that the light-saturated rate of photosystem (PS) I activity was unaffected by P or S limitation, whereas light-saturated PSII activity was reduced by more than 50%. This decline in PSII activity correlated with a decline in both the maximal quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers (PSII centers capable of performing a charge separation but unable to reduce the plastoquinone pool). In addition to a decline in the light-saturated rate of O2 evolution, there was reduced efficiency of excitation energy transfer to the reaction centers of PSII (because of dissipation of absorbed light energy as heat and because of a transition to state 2). These findings establish a common suite of alterations in photosynthetic electron transport that results in decreased linear electron flow when C. reinhardtii is limited for either P or S. It was interesting that the decline in the maximum quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers were regulated specifically during S-limited growth by the SacI gene product, which was previously shown to be critical for the acclimation of C. reinhardtii to S limitation (J.P. Davies, F.H. Yildiz, and A.R. Grossman [1996] EMBO J 15: 2150–2159).

In Vitro Reconstitution of Electron Transport from Glucose-6-Phosphate and NADPH to Nitrite1

An NADPH-dependent NO2−-reducing system was reconstituted in vitro using ferredoxin (Fd) NADP+ oxidoreductase (FNR), Fd, and nitrite reductase (NiR) from the green alga Chlamydomonas reinhardtii. NO2− reduction was dependent on all protein components and was operated under either aerobic or anaerobic conditions. NO2− reduction by this in vitro pathway was inhibited up to 63% by 1 mm NADP+. NADP+ did not affect either methyl viologen-NiR or Fd-NiR activity, indicating that inhibition was mediated through FNR. When NADPH was replaced with a glucose-6-phosphate dehydrogenase (G6PDH)-dependent NADPH-generating system, rates of NO2− reduction reached approximately 10 times that of the NADPH-dependent system. G6PDH could be replaced by either 6-phosphogluconate dehydrogenase or isocitrate dehydrogenase, indicating that G6PDH functioned to: (a) regenerate NADPH to support NO2− reduction and (b) consume NADP+, releasing FNR from NADP+ inhibition. These results demonstrate the ability of FNR to facilitate the transfer of reducing power from NADPH to Fd in the direction opposite to that which occurs in photosynthesis. The rate of G6PDH-dependent NO2− reduction observed in vitro is capable of accounting for the observed rates of dark NO3− assimilation by C. reinhardtii.

Structure and conformational changes of DNA topoisomerase II visualized by electron microscopy.

Type II DNA topoisomerases, which create a transient gate in duplex DNA and transfer a second duplex DNA through this gate, are essential for topological transformations of DNA in prokaryotic and eukaryotic cells and are of interest not only from a mechanistic perspective but also because they are targets of agents for anticancer and antimicrobial chemotherapy. Here we describe the structure of the molecule of human topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] as seen by scanning transmission electron microscopy. A globular approximately 90-angstrom diameter core is connected by linkers to two approximately 50-angstrom domains, which were shown by comparison with genetically truncated Saccharomyces cerevisiae topoisomerase II to contain the N-terminal region of the approximately 170-kDa subunits and that are seen in different orientations. When the ATP-binding site is occupied by a nonhydrolyzable ATP analog, a quite different structure is seen that results from a major conformational change and consists of two domains approximately 90 angstrom and approximately 60 angstrom in diameter connected by a linker, and in which the N-terminal domains have interacted. About two-thirds of the molecules show an approximately 25 A tunnel in the apical part of the large domain, and the remainder contain an internal cavity approximately 30 A wide in the large domain close to the linker region. We propose that structural rearrangements lead to this displacement of an internal tunnel. The tunnel is likely to represent the channel through which one DNA duplex, after capture in the clamp formed by the N-terminal domains, is transferred across the interface between the enzyme's subunits. These images are consistent with biochemical observations and provide a structural basis for understanding the reaction of topoisomerase II.

A multimer model for P680, the primary electron donor of photosystem II.

We consider a model of the photosystem II (PS II) reaction center in which its spectral properties result from weak (approximately 100 cm-1) excitonic interactions between the majority of reaction center chlorins. Such a model is consistent with a structure similar to that of the reaction center of purple bacteria but with a reduced coupling of the chlorophyll special pair. We find that this model is consistent with many experimental studies of PS II. The similarity in magnitude of the exciton coupling and energetic disorder in PS II results in the exciton states being structurally highly heterogeneous. This model suggests that P680, the primary electron donor of PS II, should not be considered a dimer but a multimer of several weakly coupled pigments, including the pheophytin electron acceptor. We thus conclude that even if the reaction center of PS II is structurally similar to that of purple bacteria, its spectroscopy and primary photochemistry may be very different.

The electron-furfural scattering dynamics for 63 energetically open electronic states

We report on integral-, momentum transfer-and differential cross sections for elastic and electronically inelastic electron collisions with furfural (C5H4O2). The calculations were performed with two different theoretical methodologies, the Schwinger multichannel method with pseudopotentials (SMCPP) and the independent atom method with screening corrected additivity rule (IAM-SCAR) that now incorporates a further interference (I) term. The SMCPP with N energetically open electronic states (N-open) at either the static-exchange (N-open ch-SE) or the static-exchange-plus-polarisation (N-open ch-SEP) approximation was employed to calculate the scattering amplitudes at impact energies lying between 5 eV and 50 eV, using a channel coupling scheme that ranges from the 1ch-SEP up to the 63ch-SE level of approximation depending on the energy considered. For elastic scattering, we found very good overall agreement at higher energies among our SMCPP cross sections, our IAM-SCAR+I cross sections and the experimental data for furan (a molecule that differs from furfural only by the substitution of a hydrogen atom in furan with an aldehyde functional group). This is a good indication that our elastic cross sections are converged with respect to the multichannel coupling effect for most of the investigated intermediate energies. However, although the present application represents the most sophisticated calculation performed with the SMCPP method thus far, the inelastic cross sections, even for the low lying energy states, are still not completely converged for intermediate and higher energies. We discuss possible reasons leading to this discrepancy and point out what further steps need to be undertaken in order to improve the agreement between the calculated and measured cross sections. (C) 2016 AIP Publishing LLC.

Optical spin transfer in ferromagnetic semiconductors

Circularly polarized laser pulses that excite electron-hole pairs across the band gap of (III,Mn)V ferromagnetic semiconductors can be used to manipulate and to study collective magnetization dynamics. The initial spin orientation of a photocarrier in a (III,V) semiconductors is determined by the polarization state of the laser. We show that the photocarrier spin can be irreversibly transferred to the collective magnetization, whose dynamics can consequently be flexibly controlled by suitably chosen laser pulses. As illustrations we demonstrate the feasibility of all optical ferromagnetic resonance and optical magnetization reorientation.

Inelastic electron tunneling spectroscopy of a Mn dimer

A scanning tunneling microscope can probe the inelastic spin excitations of single magnetic atoms in a surface via spin-flip assisted tunneling. A particular and intriguing case is the Mn dimer case. We show here that the existing theories for inelastic transport spectroscopy do not explain the observed spin transitions when both atoms are equally coupled to the scanning tunneling microscope tip and the substrate, the most likely experimental situation. The hyperfine coupling to the nuclear spins is shown to lead to a finite excitation amplitude, but the physical mechanism leading to the large inelastic signal observed is still unknown. We discuss some other alternatives that break the symmetry of the system and allow for larger excitation probabilities.

A systematic study on the influence of carbon on the behavior of hard-to-ionize elements in inductively coupled plasma–mass spectrometry

A systematic study on the influence of carbon on the signal of a large number of hard-to-ionize elements (i.e. B, Be, P, S, Zn, As, Se, Pd, Cd, Sb, I, Te, Os, Ir, Pt, Au, and Hg) in inductively coupled plasma–mass spectrometry has been carried out. To this end, carbon matrix effects have been evaluated considering different plasma parameters (i.e. nebulizer gas flow rate, r.f. power and sample uptake rate), sample introduction systems, concentration and type of carbon matrix (i.e. glycerol, citric acid, potassium citrate and ammonium carbonate) and type of mass spectrometer (i.e. quadrupole filter vs. double-focusing sector field mass spectrometer). Experimental results show that P, As, Se, Sb, Te, I, Au and Hg sensitivities are always higher for carbon-containing solutions than those obtained without carbon. The other hard-to-ionize elements (Be, B, S, Zn, Pd, Cd, Os, Ir and Pt) show no matrix effect, signal enhancement or signal suppression depending on the experimental conditions selected. The matrix effects caused by the presence of carbon are explained by changes in the plasma characteristics and the corresponding changes in ion distribution in the plasma (as reflected in the signal behavior plot, i.e. the signal intensity as a function of the nebulizer gas flow rate). However, the matrix effects for P, As, Se, Sb, Te, I, Au and Hg are also related to an increase in analyte ion population caused as a result of charge transfer reactions involving carbon-containing charged species in the plasma. The predominant specie is C+, but other species such as CO+, CO2+, C2+ and ArC+ could also play a role. Theoretical data suggest that B, Be, S, Pd, Cd, Os, Ir and Pt could also be involved in carbon based charge transfer reactions, but no experimental evidence substantiating this view has been found.

Evidence of Intracrystalline Mesostructured Porosity in Zeolites by Advanced Gas Sorption, Electron Tomography and Rotation Electron Diffraction

The small size of micropores (typically <1 nm) in zeolites causes slow diffusion of reactant and product molecules in and out of the pores and negatively impacts the product selectivity of zeolite based catalysts, for example, fluid catalytic cracking (FCC) catalysts. Size-tailored mesoporosity was introduced into commercial zeolite Y crystals by a simple surfactant-templating post-synthetic mesostructuring process. The resulting mesoporous zeolite Y showed significantly improved product selectivity in both laboratory testing and refinery trials. Advanced characterization techniques such as electron tomography, three-dimensional rotation electron diffraction, and high resolution gas adsorption coupled with hysteresis scanning and density functional theory, unambiguously revealed the intracystalline nature and connectivity of the introduced mesopores. They can be considered as molecular highways that help reactant and product molecules diffuse quickly to and away from the catalytically active sites within the zeolite crystals and, thus, shift the selectivity to favor the production of more of the valuable liquid fuels at reduced yields of coke and unconverted feed.