Particles move along actin filament bundles in nerve growth cones.

Organelle movement along actin filaments has been demonstrated in dissociated squid axoplasm [Kurznetsov, S. A., Langford, G.M. & Weiss, D. G. (1992) Nature (London) 356, 722-725 and Bearer, E.L., DeGiorgis, J.A., Bodner, R.A., Kao, A.W. & Reese, T.S. (1993) Proc. Natl. Acad. Sci. USA 90, 11252-11256] but has not been shown to occur in intact neurons. Here we demonstrate that intracellular transport occurs along actin filament bundles in intact neuronal growth cones. We used video-enhanced differential interference contrast microscopy to observe intracellular transport in superior cervical ganglion neurons cultured under conditions that enhance the visibility of actin bundles within growth cone lamellipodia. Intracellular particles, ranging in size from < 0.5-1.5 microns, moved along linear structures (termed transport bundles) at an average maximum rate of 0.48 micron/sec. After particle movement had been viewed, cultures were preserved by rapid perfusion with chemical fixative. To determine whether particle transport occurred along actin, we then used fluorescence microscopy to correlate this movement with actin and microtubule distributions in the same growth cones. The observed transport bundles colocalized with actin but not with microtubules. The rates of particle movement and the association of moving particles with actin filament bundles suggest that myosins may participate in the transport of organelles (or other materials) in intact neurons.

Efficient studies of long-distance Bmp5 gene regulation using bacterial artificial chromosomes

The regulatory regions surrounding many genes may be large and difficult to study using standard transgenic approaches. Here we describe the use of bacterial artificial chromosome clones to rapidly survey hundreds of kilobases of DNA for potential regulatory sequences surrounding the mouse bone morphogenetic protein-5 (Bmp5) gene. Simple coinjection of large insert clones with lacZ reporter constructs recapitulates all of the sites of expression observed previously with numerous small constructs covering a large, complex regulatory region. The coinjection approach has made it possible to rapidly survey other regions of the Bmp5 gene for potential control elements, to confirm the location of several elements predicted from previous expression studies using regulatory mutations at the Bmp5 locus, to test whether Bmp5 control regions act similarly on endogenous and foreign promoters, and to show that Bmp5 control elements are capable of rescuing phenotypic effects of a Bmp5 deficiency. This rapid approach has identified new Bmp5 control regions responsible for controlling the development of specific anatomical structures in the vertebrate skeleton. A similar approach may be useful for studying complex control regions surrounding many other genes important in embryonic development and human disease.

Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion

Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here, we present the crystal structure of the dimeric (Mr, 290,000) bovine milk XDH at 2.1-Å resolution and XO at 2.5-Å resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423—Lys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme.

Z-membranes: artificial organelles for overexpressing recombinant integral membrane proteins.

We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of beta-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 micrometers in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes.

How are close residues of protein structures distributed in primary sequence?

Structurally neighboring residues are categorized according to their separation in the primary sequence as proximal (1-4 positions apart) and otherwise distal, which in turn is divided into near (5-20 positions), far (21-50 positions), very far ( > 50 positions), and interchain (from different chains of the same structure). These categories describe the linear distance histogram (LDH) for three-dimensional neighboring residue types. Among the main results are the following: (i) nearest-neighbor hydrophobic residues tend to be increasingly distally separated in the linear sequence, thus most often connecting distinct secondary structure units. (ii) The LDHs of oppositely charged nearest-neighbors emphasize proximal positions with a subsidiary maximum for very far positions. (iii) Cysteine-cysteine structural interactions rarely involve proximal positions. (iv) The greatest numbers of interchain specific nearest-neighbors in protein structures are composed of oppositely charged residues. (v) The largest fraction of side-chain neighboring residues from beta-strands involves near positions, emphasizing associations between consecutive strands. (vi) Exposed residue pairs are predominantly located in proximal linear positions, while buried residue pairs principally correspond to far or very far distal positions. The results are principally invariant to protein sizes, amino acid usages, linear distance normalizations, and over- and underrepresentations among nearest-neighbor types. Interpretations and hypotheses concerning the LDHs, particularly those of hydrophobic and charged pairings, are discussed with respect to protein stability and functionality. The pronounced occurrence of oppositely charged interchain contacts is consistent with many observations on protein complexes where multichain stabilization is facilitated by electrostatic interactions.

Trapping of branched DNA in microfabricated structures.

We have observed electrostatic trapping of tribranched DNA molecules undergoing electrophoresis in a microfabricated pseudo-two-dimensional array of posts. Trapping occurs in a unique transport regimen in which the electrophoretic mobility is extremely sensitive to polymer topology. The arrest of branched polymers is explained by considering their center-of-mass motion; in certain conformations, owing to the constraints imposed by the obstacles a molecule cannot advance without the center of mass first moving a short distance backwards. The depth of the resulting local potential well can be much greater than the thermal energy so that escape of an immobilized molecule can be extremely slow. We summarize the expected behavior of the mobility as a function of field strength and topology and point out that the microfabricated arrays are highly suitable for detecting an extremely small number of branched molecules in a very large population of linear molecules.