Identification of interfaces involved in weak interactions with application to F-actin-aldolase rafts

Macromolecular interactions occur with widely varying affinities. Strong interactions form well defined interfaces but weak interactions are more dynamic and variable. Weak interactions can collectively lead to large structures such as microvilli via cooperativity and are often the precursors of much stronger interactions, e.g. the initial actin-myosin interaction during muscle contraction. Electron tomography combined with subvolume alignment and classification is an ideal method for the study of weak interactions because a 3-D image is obtained for the individual interactions, which subsequently are characterized collectively. Here we describe a method to characterize heterogeneous F-actin-aldolase interactions in 2-D rafts using electron tomography. By forming separate averages of the two constituents and fitting an atomic structure to each average, together with the alignment information which relates the raw motif to the average, an atomic model of each crosslink is determined and a frequency map of contact residues is computed. The approach should be applicable to any large structure composed of constituents that interact weakly and heterogeneously.     

POU-domain gene expression in the gastrointestinal tract

The process of self-renewal which occurs in the gastrointestinal epithelium is greatly amplified and accelerated during the intestinal adaptation which occurs in the residual ileum after massive small bowel resection (MSBR). As with growth and development, these processes must involve the coordinated regulation of many genes. Several families of nuclear proteins are known to be involved in the control of gene expression during development including the POU-domain genes; their expression has not been characterized in the gastrointestinal tract during normal cellular renewal or adaptation, and POU-domain encoding cDNAs were cloned from ileal RNA. Three known genes were cloned: Oct-1, Brn-1 and Tst-1 but no novel members of this gene family were identified. The encoded sequence for rat Oct-1 differs from that previously reported. Oct-1 is relatively ubiquitously expressed with increased expression during both development and adaptation. Minimal expression of Tst-1 was observed. Brn-1 exhibits limited expression in the adult gastrointestinal tract. but may play a role in the fetal gastrointestinal tract.     

Distribution and abundance of young fish in the St. Clair River and associated waters,Ontario

Fish larvae were sampled in 1986 in the St. Clair River, and adjacent waters. Species richness (9 taxa as larvae; 4 others as juveniles) and abundance was lowest in the river, where many larvae (e.g., burbot, rainbow smelt, and yellow perch) were in transit from Lake Huron. The most abundant, and localized, species was gizzard shad, which reached a peak mean density of 4600 larvae 100 m^-3 in an agricultural canal. Adjacent waters contribute greatly to the fish communities of the river and adjoining Lakes Huron and Erie, especially in terms of the number and quantity of forage species.     

Drosophila Bcl-2 proteins participate in stress-induced apoptosis, but are not required for normal development

Many developing tissues require programmed cell death (PCD) for proper formation. In mice and C. elegans, developmental PCD is regulated by the Bcl-2 family of proteins. Two bcl-2 genes are encoded in the Drosophila genome (debcl/dBorg1/Drob-1/dBok and buffy/dBorg2) and previous RNAi-based studies suggested a requirement for these in embryonic development. However, we report here that, despite the fact that many tissues in fruit flies are shaped by PCD, deletion of the bcl-2 genes does not perturb normal development. We investigated whether the fly bcl-2 genes regulate non-apoptotic processes that require caspases, but found these to be bcl-2 gene-independent. However, irradiation of the mutants demonstrates that DNA damage-induced apoptosis, mediated by Reaper, is blocked by buffy and that debcl is required to inhibit buffy. Our results demonstrate that developmental PCD regulation in the fly does not rely upon the Bcl-2 proteins, but that they provide an added layer of protection in the apoptotic response to stress.     

Phosphorylated smooth muscle heavy meromyosin shows an open conformation linked to activation

Smooth muscle myosin and smooth muscle heavy meromyosin (smHMM) are activated by regulatory light chain phosphorylation, but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions between motor domains. The "free head" can bind to actin, but the actin binding interface of the "blocked head" is involved in interactions with the free head. We report here a three-dimensional structure for phosphorylated, active smHMM obtained using electron crystallography of two-dimensional arrays. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state but occur between different molecules, not within the same molecule. The light chain binding domain structure of phosphorylated smHMM differs markedly from that of the "blocked" head of dephosphorylated smHMM. We hypothesize that regulatory light chain phosphorylation opens the inhibited conformation primarily by its effect on the blocked head. Singly phosphorylated smHMM is not compatible with the closed conformation if the blocked head is phosphorylated. This concept has implications for the extent of myosin activation at low levels of phosphorylation in smooth muscle.