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.

Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography

The positional relationships among all of the visible organelles in a densely packed region of cytoplasm from an insulin secreting, cultured mammalian cell have been analyzed in three dimensions (3-D) at ≈6 nm resolution. Part of a fast frozen/freeze-substituted HIT-T15 cell that included a large portion of the Golgi ribbon was reconstructed in 3-D by electron tomography. The reconstructed volume (3.1 × 3.2 × 1.2 μm3) allowed sites of interaction between organelles, and between microtubules and organellar membranes, to be accurately defined in 3-D and quantitatively analyzed by spatial density analyses. Our data confirm that the Golgi in an interphase mammalian cell is a single, ribbon-like organelle composed of stacks of flattened cisternae punctuated by openings of various sizes [Rambourg, A., Clermont, Y., & Hermo, L. (1979) Am. J. Anat. 154, 455–476]. The data also show that the endoplasmic reticulum (ER) is a single continuous compartment that forms close contacts with mitochondria, multiple trans Golgi cisternae, and compartments of the endo-lysosomal system. This ER traverses the Golgi ribbon from one side to the other via cisternal openings. Microtubules form close, non-random associations with the cis Golgi, the ER, and endo-lysosomal compartments. Despite the dense packing of organelles in this Golgi region, ≈66% of the reconstructed volume is calculated to represent cytoplasmic matrix. We relate the intimacy of structural associations between organelles in the Golgi region, as quantified by spatial density analyses, to biochemical mechanisms for membrane trafficking and organellar communication in mammalian cells.

Purified inositol hexakisphosphate kinase is an ATP synthase: diphosphoinositol pentakisphosphate as a high-energy phosphate donor.

Diphosphoinositol pentakisphosphate (PP-IP5) and bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) are recently identified inositol phosphates that possess pyrophosphate bonds. We have purified an inositol hexakisphosphate (IP6) kinase from rat brain supernatants. The pure protein, a monomer of 54 kDa, displays high affinity (Km = 0.7 microM) and selectivity for inositol hexakisphosphate as substrate. It can be dissociated from bis(diphospho)inositol tetrakisphosphate synthetic activity. The purified enzyme transfers a phosphate from PP-IP5 to ADP to form ATP. This ATP synthase activity indicates the high phosphate group transfer potential of PP-IP5 and may represent a physiological role for PP-IP5.

Ultrafast diffraction and structural dynamics: The nature of complex molecules far from equilibrium

Studies of molecular structures at or near their equilibrium configurations have long provided information on their geometry in terms of bond distances and angles. Far-from-equilibrium structures are relatively unknown—especially for complex systems—and generally, neither their dynamics nor their average geometries can be extrapolated from equilibrium values. For such nonequilibrium structures, vibrational amplitudes and bond distances play a central role in phenomena such as energy redistribution and chemical reactivity. Ultrafast electron diffraction, which was developed to study transient molecular structures, provides a direct method for probing the nature of complex molecules far from equilibrium. Here we present our ultrafast electron diffraction observations of transient structures for two cyclic hydrocarbons. At high internal energies of ≈4 eV, these molecules display markedly different behavior. For 1,3,5-cycloheptatriene, excitation results in the formation of hot ground-state structures with bond distances similar to those of the initial structure, but with nearly three times the average vibrational amplitude. Energy is redistributed within 5 ps, but with a negative temperature characterizing the nonequilibrium population. In contrast, the ring-opening reaction of 1,3-cyclohexadiene is shown to result in hot structures with a C—C bond distance of over 1.7 Å, which is 0.2 Å away from any expected equilibrium value. Even up to 400 ps, energy remains trapped in large-amplitude motions comprised of torsion and asymmetric stretching. These studies promise a new direction for studying structural dynamics in nonequilibrium complex systems.

Overexpression of human copper,zinc-superoxide dismutase (SOD1) prevents postischemic injury

Superoxide and superoxide-derived oxidants have been hypothesized to be important mediators of postischemic injury. Whereas copper,zinc-superoxide dismutase, SOD1, efficiently dismutates superoxide, there has been controversy regarding whether increasing intracellular SOD1 expression would protect against or potentiate cellular injury. To determine whether increased SOD1 protects the heart from ischemia and reperfusion, studies were performed in a newly developed transgenic mouse model in which direct measurement of superoxide, contractile function, bioenergetics, and cell death could be performed. Transgenic mice with overexpression of human SOD1 were studied along with matched nontransgenic controls. Immunoblotting and immunohistology demonstrated that total SOD1 expression was increased 10-fold in hearts from transgenic mice compared with nontransgenic controls, with increased expression in both myocytes and endothelial cells. In nontransgenic hearts following 30 min of global ischemia a reperfusion-associated burst of superoxide generation was demonstrated by electron paramagnetic resonance spin trapping. However, in the transgenic hearts with overexpression of SOD1 the burst of superoxide generation was almost totally quenched, and this was accompanied by a 2-fold increase in the recovery of contractile function, a 2.2-fold decrease in infarct size, and a greatly improved recovery of high energy phosphates compared with that in nontransgenic controls. These results demonstrate that superoxide is an important mediator of postischemic injury and that increasing intracellular SOD1 dramatically protects the heart from this injury. Thus, increasing intracellular SOD1 expression may be a highly effective approach to decrease the cellular injury that occurs following reperfusion of ischemic tissues.

Implications of a possible clustering of highest-energy cosmic rays

Recently, a possible clustering of a subset of observed ultra-high energy cosmic rays above ≃40 EeV (4 × 1019 eV) in pairs near the supergalactic plane was reported. We show that a confirmation of this effect would provide information on the origin and nature of these events and, in case of charged primaries, imply interesting constraints on the extragalactic magnetic field. Possible implications for the most common models of ultra-high energy cosmic ray production in the literature are discussed.

Silver-based crystalline nanoparticles, microbially fabricated

One mechanism of silver resistance in microorganisms is accumulation of the metal ions in the cell. Here, we report on the phenomenon of biosynthesis of silver-based single crystals with well-defined compositions and shapes, such as equilateral triangles and hexagons, in Pseudomonas stutzeri AG259. The crystals were up to 200 nm in size and were often located at the cell poles. Transmission electron microscopy, quantitative energy-dispersive x-ray analysis, and electron diffraction established that the crystals comprise at least three different types, found both in whole cells and thin sections. These Ag-containing crystals are embedded in the organic matrix of the bacteria. Their possible potential as organic-metal composites in thin film and surface coating technology is discussed.

Field theory in superfluid 3He: What are the lessons for particle physics, gravity, and high-temperature superconductivity?

There are several classes of homogeneous Fermi systems that are characterized by the topology of the energy spectrum of fermionic quasiparticles: (i) gapless systems with a Fermi surface, (ii) systems with a gap in their spectrum, (iii) gapless systems with topologically stable point nodes (Fermi points), and (iv) gapless systems with topologically unstable lines of nodes (Fermi lines). Superfluid 3He-A and electroweak vacuum belong to the universality class 3. The fermionic quasiparticles (particles) in this class are chiral: they are left-handed or right-handed. The collective bosonic modes of systems of class 3 are the effective gauge and gravitational fields. The great advantage of superfluid 3He-A is that we can perform experiments by using this condensed matter and thereby simulate many phenomena in high energy physics, including axial anomaly, baryoproduction, and magnetogenesis. 3He-A textures induce a nontrivial effective metrics of the space, where the free quasiparticles move along geodesics. With 3He-A one can simulate event horizons, Hawking radiation, rotating vacuum, etc. High-temperature superconductors are believed to belong to class 4. They have gapless fermionic quasiparticles with a “relativistic” spectrum close to gap nodes, which allows application of ideas developed for superfluid 3He-A.

Visualization of a water-selective pore by electron crystallography in vitreous ice

The water-selective pathway through the aquaporin-1 membrane channel has been visualized by fitting an atomic model to a 3.7-Å resolution three-dimensional density map. This map was determined by analyzing images and electron diffraction patterns of lipid-reconstituted two-dimensional crystals of aquaporin-1 preserved in vitrified buffer in the absence of any additive. The aqueous pathway is characterized by a size-selective pore that is ≈4.0 ± 0.5Å in diameter, spans a length of ≈18Å, and bends by ≈25° as it traverses the bilayer. This narrow pore is connected by wide, funnel-shaped openings at the extracellular and cytoplasmic faces. The size-selective pore is outlined mostly by hydrophobic residues, resulting in a relatively inert pathway conducive to diffusion-limited water flow. The apex of the curved pore is close to the locations of the in-plane pseudo-2-fold symmetry axis that relates the N- and C-terminal halves and the conserved, functionally important N76 and N192 residues.

A quantitative model for membrane fusion based on low-energy intermediates

The energetics of a fusion pathway is considered, starting from the contact site where two apposed membranes each locally protrude (as “nipples”) toward each other. The equilibrium distance between the tips of the two nipples is determined by a balance of physical forces: repulsion caused by hydration and attraction generated by fusion proteins. The energy to create the initial stalk, caused by bending of cis monolayer leaflets, is much less when the stalk forms between nipples rather than parallel flat membranes. The stalk cannot, however, expand by bending deformations alone, because this would necessitate the creation of a hydrophobic void of prohibitively high energy. But small movements of the lipids out of the plane of their monolayers allow transformation of the stalk into a modified stalk. This intermediate, not previously considered, is a low-energy structure that can reconfigure into a fusion pore via an additional intermediate, the prepore. The lipids of this latter structure are oriented as in a fusion pore, but the bilayer is locally compressed. All membrane rearrangements occur in a discrete local region without creation of an extended hemifusion diaphragm. Importantly, all steps of the proposed pathway are energetically feasible.

Direct structure analysis in protein electron crystallography: crystallographic phases for halorhodopsin to 6-A resolution.

The crystal structure of halorhodopsin was determined in (centrosymmetric) projection to 6-A resolution by direct methods that use only the amplitudes of the electron diffraction pattern. A multisolution technique was used to generate initial 15-A-resolution basis sets, and after selection of the best phase set (by the closest match of magnitude of Eobs and magnitude of Ecalc), annealing of individual reflections was used to improve its accuracy. The Sayre equation was then used to expand the phase terms to 10 A, followed again by phase annealing. A final expansion with the Sayre equation enlarged this corrected phase set to 6 A. When the condition of density flatness was used to locate the best phase solution after each extension, a final structure could be observed that was quite similar to the one found earlier by analysis of electron micrographs.

DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions.

DNA-strand exchange promoted by Escherichia coli RecA protein normally requires the presence of ATP and is accompanied by ATP hydrolysis, thereby implying a need for ATP hydrolysis. Previously, ATP hydrolysis was shown not to be required; here we demonstrate furthermore that a nucleoside triphosphate cofactor is not required for DNA-strand exchange. A gratuitous allosteric effector consisting of the noncovalent complex of ADP and aluminum fluoride, ADP.AIF4-, can both induce the high-affinity DNA-binding state of RecA protein and support the homologous pairing and exchange of up to 800-900 bp of DNA. These results demonstrate that induction of the functionally active, high-affinity DNA-binding state of RecA protein is needed for RecA protein-promoted DNA-strand exchange and that there is no requirement for a high-energy nucleotide cofactor for the exchange of DNA strands. Consequently, the free energy needed to activate the DNA substrates for DNA-strand exchange is not derived from ATP hydrolysis. Instead, the needed free energy is derived from ligand binding and is transduced to the DNA via the associated ligand-induced structural transitions of the RecA protein-DNA complex; ATP hydrolysis simply destroys the effector ligand. This concept has general applicability to the mechanism of energy transduction by proteins.