Cooperative and Noncooperative Assembly of Oligopyrenotides Resolved by Atomic Force Microscopy

The supramolecular assembly of amphiphilic oligopyrenotide building blocks (covalently linked heptapyrene, Py7) is studied by atomic force microscopy (AFM) in combination with optical spectroscopy. The assembly process is triggered in a controlled manner by increasing the ionic strength of the aqueous oligomer solution. Cooperative noncovalent interactions between individual oligomeric units lead to the formation of DNA-like supramolecular polymers. We also show that the terminal attachment of a single cytidine nucleotide to the heptapyrenotide (Py7-C) changes the association process from a cooperative (nucleation−elongation) to a noncooperative (isodesmic) regime, suggesting a structure misfit between the cytidine and the pyrene units. We also demonstrate that AFM enables the identification and characterization of minute concentrations of the supramolecular products, which was not accessible by conventional optical spectroscopy.

A second generation radiation hybrid map to aid the assembly of the bovine genome sequence

BACKGROUND: Several approaches can be used to determine the order of loci on chromosomes and hence develop maps of the genome. However, all mapping approaches are prone to errors either arising from technical deficiencies or lack of statistical support to distinguish between alternative orders of loci. The accuracy of the genome maps could be improved, in principle, if information from different sources was combined to produce integrated maps. The publicly available bovine genomic sequence assembly with 6x coverage (Btau_2.0) is based on whole genome shotgun sequence data and limited mapping data however, it is recognised that this assembly is a draft that contains errors. Correcting the sequence assembly requires extensive additional mapping information to improve the reliability of the ordering of sequence scaffolds on chromosomes. The radiation hybrid (RH) map described here has been contributed to the international sequencing project to aid this process. RESULTS: An RH map for the 30 bovine chromosomes is presented. The map was built using the Roslin 3000-rad RH panel (BovGen RH map) and contains 3966 markers including 2473 new loci in addition to 262 amplified fragment-length polymorphisms (AFLP) and 1231 markers previously published with the first generation RH map. Sequences of the mapped loci were aligned with published bovine genome maps to identify inconsistencies. In addition to differences in the order of loci, several cases were observed where the chromosomal assignment of loci differed between maps. All the chromosome maps were aligned with the current 6x bovine assembly (Btau_2.0) and 2898 loci were unambiguously located in the bovine sequence. The order of loci on the RH map for BTA 5, 7, 16, 22, 25 and 29 differed substantially from the assembled bovine sequence. From the 2898 loci unambiguously identified in the bovine sequence assembly, 131 mapped to different chromosomes in the BovGen RH map. CONCLUSION: Alignment of the BovGen RH map with other published RH and genetic maps showed higher consistency in marker order and chromosome assignment than with the current 6x sequence assembly. This suggests that the bovine sequence assembly could be significantly improved by incorporating additional independent mapping information.

Self-assembly of sensory neurons into ganglia-like microtissues

Unraveling intra- and inter-cellular signaling networks managing cell-fate control, coordinating complex differentiation regulatory circuits and shaping tissues and organs in living systems remain major challenges in the post-genomic era. Resting on the laurels of past-century monolayer culture technologies, the cell culture community has only recently begun to appreciate the potential of three-dimensional mammalian cell culture systems to reveal the full scope of mechanisms orchestrating the tissue-like cell quorum in space and time. Capitalizing on gravity-enforced self-assembly of monodispersed primary embryonic mouse cells in hanging drops, we designed and characterized a three-dimensional cell culture model for ganglion-like structures. Within 24h, a mixture of mouse embryonic fibroblasts (MEF) and cells, derived from the dorsal root ganglion (DRG) (sensory neurons and Schwann cells) grown in hanging drops, assembled to coherent spherical microtissues characterized by a MEF feeder core and a peripheral layer of DRG-derived cells. In a time-dependent manner, sensory neurons formed a polar ganglion-like cap structure, which coordinated guided axonal outgrowth and innervation of the distal pole of the MEF feeder spheroid. Schwann cells, present in embryonic DRG isolates, tended to align along axonal structures and myelinate them in an in vivo-like manner. Whenever cultivation exceeded 10 days, DRG:MEF-based microtissues disintegrated due to an as yet unknown mechanism. Using a transgenic MEF feeder spheroid, engineered for gaseous acetaldehyde-inducible interferon-beta (ifn-beta) production by cotransduction of retro-/ lenti-viral particles, a short 6-h ifn-beta induction was sufficient to rescue the integrity of DRG:MEF spheroids and enable long-term cultivation of these microtissues. In hanging drops, such microtissues fused to higher-order macrotissue-like structures, which may pave the way for sophisticated bottom-up tissue engineering strategies. DRG:MEF-based artificial micro- and macrotissue design demonstrated accurate key morphological aspects of ganglions and exemplified the potential of self-assembled scaffold-free multicellular micro-/macrotissues to provide new insight into organogenesis.

Adenine nucleotide-dependent regulation of assembly of bacterial tubulin-like FtsZ by a hypermorph of bacterial actin-like FtsA.

Cytokinesis in bacteria depends upon the contractile Z ring, which is composed of dynamic polymers of the tubulin homolog FtsZ as well as other membrane-associated proteins such as FtsA, a homolog of actin that is required for membrane attachment of the Z ring and its subsequent constriction. Here we show that a previously characterized hypermorphic mutant FtsA (FtsA*) partially disassembled FtsZ polymers in vitro. This effect was strictly dependent on ATP or ADP binding to FtsA* and occurred at substoichiometric levels relative to FtsZ, similar to cellular levels. Nucleotide-bound FtsA* did not affect FtsZ GTPase activity or the critical concentration for FtsZ assembly but was able to disassemble preformed FtsZ polymers, suggesting that FtsA* acts on FtsZ polymers. Microscopic examination of the inhibited FtsZ polymers revealed a transition from long, straight polymers and polymer bundles to mainly short, curved protofilaments. These results indicate that a bacterial actin, when activated by adenine nucleotides, can modify the length distribution of bacterial tubulin polymers, analogous to the effects of actin-depolymerizing factor/cofilin on F-actin.

The C-terminal domain of MinC inhibits assembly of the Z ring in Escherichia coli.

In Escherichia coli, the Min system, consisting of three proteins, MinC, MinD, and MinE, negatively regulates FtsZ assembly at the cell poles, helping to ensure that the Z ring will assemble only at midcell. Of the three Min proteins, MinC is sufficient to inhibit Z-ring assembly. By binding to MinD, which is mostly localized at the membrane near the cell poles, MinC is sequestered away from the cell midpoint, increasing the probability of Z-ring assembly there. Previously, it has been shown that the two halves of MinC have two distinct functions. The N-terminal half is sufficient for inhibition of FtsZ assembly, whereas the C-terminal half of the protein is required for binding to MinD as well as to a component of the division septum. In this study, we discovered that overproduction of the C-terminal half of MinC (MinC(122-231)) could also inhibit cell division and that this inhibition was at the level of Z-ring disassembly and dependent on MinD. We also found that fusing green fluorescent protein to either the N-terminal end of MinC(122-231), the C terminus of full-length MinC, or the C terminus of MinC(122-231) perturbed MinC function, which may explain why cell division inhibition by MinC(122-231) was not detected previously. These results suggest that the C-terminal half of MinC has an additional function in the regulation of Z-ring assembly.

Characterization of NARJ: A molecular chaperone involved in assembly of nitrate reductase

The major goal of this work was to define the role of accessory protein, NARJ, in assembly of nitrate reductase which is a membrane-bound multisubunit enzyme that can catalyze the reduction of nitrate to nitrite under anaerobic growth in E. coli. Nitrate reductase is encoded by the nar GHJI operon under control of the narG promoter. The purified nitrate reductase is composed of three subunits: $\alpha,\ \beta,$ and $\gamma.$ The NARJ protein which is encoded by the third gene (narJ) is not found to be associated with any of the purified preparations of the enzyme, but is required for active nitrate reductase. In this study the product of the narJ gene was identified. NARJ appeared to be produced at a reduced level, compared to the other proteins encoded by the nar operon. Since NARJ could not be overexpressed to a level for an efficient purification, NARJ was expressed and purified as a recombinant protein with polyhistidine tag. The recombinant protein NARJ-6His could functionally replace native NARJ. Purified NARJ-6His is a dimeric protein which contains no identifiable cofactors or unique secondary structure. NARJ was localized in the cytoplasm, and was not associated with nitrate reductase in the membrane. In vivo NARJ activated the $\alpha\beta$ complex and stabilized the $\alpha$ subunit against protease degradation. In the absence of the membrane-bound $\gamma$ subunit, NARJ formed an intermediate complex with $\alpha\beta$ in the cytosol. Based on these studies, NARJ fits the formal definition of a molecular chaperone. It appears to be required only for the biogenesis of nitrate reductase and, therefore, is defined as a private chaperone specifically involved in the assembly of nitrate reductase system. ^