Gap junction structures. IV. Asymmetric features revealed by low- irradiation microscopy

Micrographs of mouse liver gap junctions, isolated with detergents, and negatively stained with uranyl acetate, have been recorded by low-irradiation methods. Our Fourier-averaged micrographs of the hexagonal junction lattice show skewed, hexameric connexons with less stain at the threefold axis than at the six indentations between the lobes of the connexon image. These substructural features, not clearly observed previously, are acutely sensitive to irradiation. After an electron dose less than that normally used in microscopy, the image is converted to the familiar doughnut shape, with a darkly stained center and a smooth hexagonal outline, oriented with mirror symmetry in the lattice. Differences in appearance among 25 reconstructed images from our low-irradiation micrographs illustrate variation in staining of the connexon channel and the space between connexons. Consistently observed stain concentration at six symmetrically related sites approximately 34 A from the connexon center, 8 degrees to the right or left of the (1, 1) lattice vector may reveal an intrinsic asymmetric feature of the junction structure. The unexpected skewing of the six-lobed connexon image suggests that the pair of hexagonal membrane arrays that form the junction may not be structurally identical. Because the projected image of the connexon pair itself appears mirror symmetric, each pair may consist of two identical connexon hexamers related by local (noncrystallographic) twofold axes in the junctional plane at the middle of the gap. All connexons may be chemically identical, but their packing in the hexagonal arrays on the two sides of the junction appears to be nonequivalent.     

Weighted averaging,logistic regression and the Gaussian response model

The indicator value and ecological amplitude of a species with respect to a quantitative environmental variable can be estimated from data on species occurrence and environment. A simple weighted averaging (WA) method for estimating these parameters is compared by simulation with the more elaborate method of Gaussian logistic regression (GLR), a form of the generalized linear model which fits a Gaussian-like species response curve to presence-absence data. The indicator value and the ecological amplitude are expressed by two parameters of this curve, termed the optimum and the tolerance, respectively. When a species is rare and has a narrow ecological amplitude — or when the distribution of quadrats along the environmental variable is reasonably even over the species' range, and the number of quadrats is small — then WA is shown to approach GLR in efficiency. Otherwise WA may give misleading results. GLR is therefore preferred as a practical method for summarizing species' distributions along environmental gradients. Formulas are given to calculate species optima and tolerances (with their standard errors), and a confidence interval for the optimum from the GLR output of standard statistical packages.Nomenclature follows Heukels-van der Meijden (1983).We would like to thank Drs I. C. Prentice, N. J. M. Gremmen and J. A. Hoekstra for comments on the paper. We are grateful to Ir. Th. A. de Boer (CABO, Wageningen) for permission to use the data of the first example.     

Movement and self-control in protein assemblies. Quasi-equivalence revisited.

Purposeful switching among different conformational states exerts self-control in the construction and action of protein assemblies. Quasi-equivalence, conceived to explain icosahedral virus structure, arises by differentiation of identical protein subunits into different conformations that conserve essential bonding specificity. Mechanical models designed to represent the energy distribution in the structure, rather than just the arrangement of matter, are used to explore flexibility and self-controlled movements in virus particles. Information about the assembly of bacterial flagella, actin, tobacco mosaic virus and the T4 bacteriophage tail structure show that assembly can be controlled by switching the subunits from an inactive, unsociable form to an active, associable form. Energy to drive this change is provided by the intersubunit bonding in the growing structure; this self-control of assembly by conformational switching is called "autostery", by homology with allostery. A mechanical model of the contractile T4 tail sheath has been constructed to demonstrate how self-controlled activation of a latent bonding potential can drive a purposeful movement. The gradient of quasi-equivalent conformations modelled in the contracting tail sheath has suggested a workable mechanism for self-determination of tail tube length. Concerted action by assemblies of identical proteins may often depend on individually differentiated movements.     

The symmetries of filamentous phage particles

The helical symmetries of two classes of filamentous bacteriophage particles are distinctly different. The symmetry of the class I particles is²C₅S_~2.0 (a 5-fold rotation axis combined with an approximately 2-fold screw axis). The symmetry of the class II particles is C₁S_5.4 (a one-start helix with 27 subunits equally spaced along five turns). The same basic α-helical interlocking arrangement of the largely α-helical coat protein subunits can be accommodated by the symmetry of the two classes of phage particles. The conservation of this structural pattern reflects intrinsic packing properties of α-helices. The difference between the symmetries of the class I and class II particles suggests that different assembly processes may have evolved to form these structures with very similar protein packing architectures.     

Mutants of Arabidopsis with altered regulation of starch degradation

Mutants of Arabidopsis thaliana (L.) Heynh. with altered regulation of starch degradation were identified by screening for plants that retained high levels of leaf starch after a period of extended darkness. The mutant phenotype was also expressed in seeds, flowers, and roots, indicating that the same pathway of starch degradation is used in these tissues. In many respects, the physiological consequences of the mutations were equivalent to the effects observed in previously characterized mutants of Arabidopsis that are unable to synthesize starch. One mutant line, which was characterized in detail, had normal levels of activity of the starch degradative enzymes α-amylase, β-amylase, phosphorylase, D-enzyme, and debranching enzyme. Thus, it was not possible to establish a biochemical basis for the phenotype, which was due to a recessive mutation at a locus designated sex1 at position 12.2 on chromosome 1. This raises the possibility that hitherto unidentified factors, altered by the mutation, play a key role in regulating or catalyzing starch degradation.     

Vps33B is required for delivery of endocytosed cargo to lysosomes

Lysosomes are the main degradative compartments of eukaryotic cells. The CORVET and HOPS tethering complexes are well known for their role in membrane fusion in the yeast endocytic pathway. Yeast Vps33p is part of both complexes, and has two mammalian homologues: Vps33A and Vps33B. Vps33B is required for recycling of apical proteins in polarized cells and a causative gene for ARC syndrome. Here, we investigate whether Vps33B is also required in the degradative pathway. By fluorescence and electron microscopy we show that Vps33B depletion in HeLa cells leads to significantly increased numbers of late endosomes that together with lysosomes accumulate in the perinuclear region. Degradation of endocytosed cargo is impaired in these cells. By electron microscopy we show that endocytosed BSA‐gold reaches late endosomes, but is decreased in lysosomes. The increase in late endosome numbers and the lack of internalized cargo in lysosomes are indicative for a defect in late endosomal–lysosomal fusion events, which explains the observed decrease in cargo degradation. A corresponding phenotype was found after Vps33A knock down, which in addition also resulted in decreased lysosome numbers. We conclude that Vps33B, in addition to its role in endosomal recycling, is required for late endosomal–lysosomal fusion events.      

Intracellular metabolite determination in the presence of extracellular abundance: Application to the penicillin biosynthesis pathway in Penicillium chrysogenum

Important steps in metabolic pathways are formed by the transport of substrates and products over the cell membrane. The study of in vivo transport kinetics requires accurate quantification of intra‐ and extracellular levels of the transported compounds. Especially in case of extracellular abundance, the proper determination of intracellular metabolite levels poses challenges. Efficient removal of extracellular substrates and products is therefore important not to overestimate the intracellular amounts. In this study we evaluated two different rapid sampling methods, one combined with cold filtration and the other with centrifugation, for their applicability to determine intracellular amounts of metabolites which are present in high concentrations in the extracellular medium. The filtration‐based method combines fast sampling and immediate quenching of cellular metabolism in cold methanol, with rapid and effective removal of all compounds present outside the cells by means of direct filtration and subsequent filtration‐based washing. In the centrifugation‐based method, removal of the extracellular metabolites from the cells was achieved by means of multiple centrifugation and resuspension steps with the cold quenching solution. The cold filtration method was found to be highly superior to the centrifugation method to determine intracellular amounts of metabolites related to penicillin‐G biosynthesis and allowed the quantification of compounds of which the extracellular amounts were 3–4 orders of magnitude higher than the intracellular amounts. Using this method for the first time allowed to measure the intracellular levels of the side chain precursor phenylacetic acid (PAA) and the product penicillin‐G of the penicillin biosynthesis pathway, compounds of which the transport mechanism in Penicillium chrysogenum is still far from being sufficiently understood. Biotechnol. Bioeng. 2010;107: 105–115. © 2010 Wiley Periodicals, Inc.     

Systematic screening of polyphosphate (poly P) levels in yeast mutant cells reveals strong interdependence with primary metabolism

Background  

Inorganic polyphosphate (poly P) occurs universally in all organisms from bacteria to man. It functions, for example, as a phosphate and energy store, and is involved in the activation and regulation of proteins. Despite its ubiquitous occurrence and important functions, it is unclear how poly P is synthesized or how poly P metabolism is regulated in higher eukaryotes. This work describes a systematic analysis of poly P levels in yeast knockout strains mutated in almost every non-essential gene.