Oberhauser, Andrés F.

The availability of the ratiometric Ca2+ indicator dyes, fura-2, and indo-1, and advances in digital imaging and computer technology have made it possible to detect Ca2+ changes in single cells with high temporal and spatial resolution. However, the optical properties of the conventional epifluorescence microscope do not produce a perfect image of the specimen. Instead, the observed image is a spatial low pass filtered version of the object and is contaminated with out of focus information. As a result, the image has reduced contrast and an increased depth of field. This problem is especially important for measurements of localized Ca2+ concentrations. One solution to this problem is to use a scanning confocal microscope which only detects in focus information, but this approach has several disadvantages for low light fluorescence measurements in living cells. An alternative approach is to use digital image processing and a deblurring algorithm to remove the out of focus information by using a knowledge of the point spread function of the microscope. All of these algorithms require a stack of two-dimensional images taken at different focal planes, although the "nearest neighbor deblurring" algorithm only requires one image above and below the image plane. We have used a modification of this scheme to construct a simple inverse filter, which extracts optical sections comparable to those of the nearest neighbors scheme, but without the need for adjacent image sections. We have used this "no neighbors" processing scheme to deblur images of fura-2-loaded mast cells from beige mice and generate high resolution ratiometric Ca2+ images of thin sections through the cell. The shallow depth of field of these images is demonstrated by taking pairs of images at different focal planes, 0.5-microns apart. The secretory granules, which exclude the fura-2, appear in focus in all sections and distinct changes in their size and shape can be seen in adjacent sections. In addition, we show, with the aid of model objects, how the combination of inverse filtering and ratiometric imaging corrects for some of the inherent limitations of using an inverse filter and can be used for quantitative measurements of localized Ca2+ gradients. With this technique, we can observe Ca2+ transients in narrow regions of cytosol between the secretory granules and plasma membrane that can be less than 0.5-microns wide. Moreover, these Ca2+ increases can be seen to coincide with the swelling of the secretory granules that follows exocytotic fusion.

Atomic force microscope manipulations of single polysaccharide molecules have recently expanded conformational chemistry to include force-driven transitions between the chair and boat conformers of the pyranose ring structure. We now expand these observations to include chair inversion, a common phenomenon in the conformational chemistry of six-membered ring molecules. We demonstrate that by stretching single pectin molecules (1 → 4-linked α- d -galactouronic acid polymer), we could change the pyranose ring conformation from a chair to a boat and then to an inverted chair in a clearly resolved two-step conversion: 4 C 1 ⇄ boat ⇄ 1 C 4 . The two-step extension of the distance between the glycosidic oxygen atoms O 1 and O 4 determined by atomic force microscope manipulations is corroborated by ab initio calculations of the increase in length of the residue vector O 1 O 4 on chair inversion. We postulate that this conformational change results from the torque generated by the glycosidic bonds when a force is applied to the pectin molecule. Hence, the glycosidic bonds act as mechanical levers, driving the conformational transitions of the pyranose ring. When the glycosidic bonds are equatorial (e), the torque is zero, causing no conformational change. However, when the glycosidic bond is axial (a), torque is generated, causing a rotation around C—C bonds and a conformational change. This hypothesis readily predicts the number of transitions observed in pyranose monomers with 1a-4a linkages (two), 1a-4e (one), and 1e-4e (none). Our results demonstrate single-molecule mechanochemistry with the capability of resolving complex conformational transitions.

Using patch-clamp capacitance and amperometric techniques, we have identified an exocytotic phenotype that affects the function of the fusion pore, the molecular structure that connects the lumen of a secretory vesicle with the extracellular environment during exocytosis. Direct observation of individual exocytotic events in mast cells from the ruby-eye mouse ( ru / ru ) showed a 3-fold increase in the fraction and duration of transient fusion events with respect to wild-type mice. The fraction of the total fusion events that were transient increased from 0.22 ± 0.02 (wild type) to 0.65 ± 0.02 ( ru / ru ), and the average duration of these events increased from 418 ± 32 ms (wild type) to 1207 ± 89 ms ( ru / ru ). We also show that this phenotype can reduce and delay an evoked secretory response by causing the fusion of vesicles that have been previously emptied by repeated cycles of transient fusion. The exocytotic phenotype that we describe here may be a cause of diseases like platelet storage pool deficiency and prolonged bleeding times for which the ruby-eye mouse serves as an animal model. Furthermore, the identification of the gene causing the fusion pore phenotype reported here will illuminate the molecular mechanisms regulating exocytotic fusion.