Cell Adhesion Molecule L1 in Folded (Horseshoe) and Extended Conformations

We have investigated the structure of the cell adhesion molecule L1 by electron microscopy. We were particularly interested in the conformation of the four N-terminal immunoglobulin domains, because x-ray diffraction showed that these domains are bent into a horseshoe shape in the related molecules hemolin and axonin-1. Surprisingly, rotary-shadowed specimens showed the molecules to be elongated, with no indication of the horseshoe shape. However, sedimentation data suggested that these domains of L1 were folded into a compact shape in solution; therefore, this prompted us to look at the molecules by an alternative technique, negative stain. The negative stain images showed a compact shape consistent with the expected horseshoe conformation. We speculate that in rotary shadowing the contact with the mica caused a distortion of the protein, weakening the bonds forming the horseshoe and permitting the molecule to extend. We have thus confirmed that the L1 molecule is primarily in the horseshoe conformation in solution, and we have visualized for the first time its opening into an extended conformation. Our study resolves conflicting interpretations from previous electron microscopy studies of L1.

Cytoplasmic free-Ca2+ level rises with repellents and falls with attractants in Escherichia coli chemotaxis.

Cytoplasmic free-Ca2+ levels in Escherichia coli were measured by use of the fluorescent Ca(2+)-indicator dye fura-2. Chemotactically wild-type E. coli regulated cytoplasmic free Ca2+ at approximately 100 nM when no stimuli were encountered, but changes in bacterial behavior correlated with changes in cytoplasmic free-Ca2+ concentration. For chemotactically wild-type E. coli, addition of a repellent resulted in cells tumbling and a transient increase in cytoplasmic free-Ca2+ levels. Conversely, addition of an attractant to wild-type cells caused running and produced a transient decrease in cytoplasmic free-Ca2+ levels. Studies with mutant strains showed that the chemoreceptors were required for the observed changes in cytoplasmic free-Ca2+ levels in response to chemical stimuli.