When used to probe EcoRI-digested Candida albicans DNA, the moderately repetitive sequence Ca3 generated a Southern blot hybridization pattern which included 15 to 25 bands, depending upon the strain. The pattern was stable through 400 generations in each of three independent strains but variable between most of 46 unrelated tester strains, making it a very effective probe for discrimination between strains. Computerassisted methods (Dendron) were developed for storage of Ca3 patterns in data files, calculation of similarity (SAB) values between strains based upon band positions and intensities, and generation of histograms and dendrograms based on SAB values for all strains or any subset of strains in large epidemiological studies. In testing the effectiveness of the system, it was found that (i) multiple isolates from different body locations of the same healthy individual could represent either the same strain or different strains, (ii) isolates from oral lesions of a husband and wife represented the same strain, (iii) strains isolated from the mouths of 10 healthy individuals on the same day and in the same geographical location were as dissimilar on average as the 46 unrelated tester strains, and (iv) strains isolated from seven immunocompromised patients hospitalized over a 2.5-month period in the same hospital were highly similar, indicating nosocomial origin. The apparent effectiveness of these fingerprinting methods and the Dendron program suggests that interlaboratory procedures for fingerprinting should be standardized and all patterns should be analyzed and stored in a common and accessible data base for broad epidemiological analysis.

Abstract. Before addition of cAMP, Dictyostelum amoebae rapidly translocating in buffer are elongate, exhibit expansion zones primarily at the anterior end and filamentous actin (F-actin) localization primarily in the anterior pseudopodia. Intracellular particle movement is primarily in the anterior direction, and the average rate of particle movement is roughly five times the rate of cellular translocation. Within seconds after the addition of 10 -6 M cAMP, there is a dramatic suppression of cellular translocation, an inhibition of pseudopod formation, a freeze in cellular morphology, a dramatic depression in intracellular particle movement, loss of F-actin localization in pseudopodia concomitant with relocalization of F-actin in the general cytoplasmic cortex under the plasma membrane, and a doubling of F-actin content. After 10 s, expansion zones are again visible at the cell perimeter, but they no longer are localized in the original anterior portion of the cell. There is a slight rebound in particle movement after 10 s, but particles with persistent tracks now show no directionality towards the original anterior portion of the cell, as they did before cAMP addition. Finally, in parallel with the resumption of peripheral expansion and the small rebound in particle movement, there is a decrease in total cellular F-actin to the untreated level. The pattern of microtubule organization is unaffected by the addition of cAMP.URING aggregation in the cellular slime mold Dictyostelium discoideum, cells in the center of an aggregation territory release the chemoattractant cAMP in a pulsatile fashion (3,30,40). Cells peripheral to the center respond by relaying the signal outwardly (6, 10) and by moving in a directed fashion towards the aggregation center (1). Because of the pulsatile nature of the original signal and the relay system, cAMP moves outwardly through the territory as a nondissipating wave. The extracellular cAMP signal is mediated by a cell surface receptor that is a member of the beta-adrenergic receptor family (15) and interacts with G proteins (16, 31).To investigate the sequence of receptor-mediated biochemical events in the cAMP response, the standard protocol has been the rapid addition of cAMP to chemotactically responsive cells suspended in or perfused with buffer. Although the increase in cAMP in the cellular environment under these experimental conditions occurs at a far faster rate than during the front of a natural wave (35, 44), a number of rapid physiological responses have been demonstrated which probably play integral roles in cAMP-mediated chemotaxis. These changes include (a) the synthesis and release of cAMP (5) Recently, the behavioral responses of cells to a rapid increase in cAMP were assessed. It was demonstrated that the rapid addition of cAMP to the peak concentration of the natural wave (10-6 M) results in an immediate decrease in velocity measured by centroid translocation (35,43,44,46), an increase in directional change (44), an increase in roundness (13,35,44), and a decrease...

We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity), and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 μm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. Images are converted to mathematical representations from which quantitative parameters can be derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named “facilitators” and “probes.” A third cell type, the “dervish”, is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.

Amebae of Dictyostelium discoideum normally chemotax to aggregation centers by assessing the direction of outwardly moving, nondissipating waves of the chemoattractant cAMP. However, D. discoideum amebae can also assess the direction of a relatively stable spatial gradient. We demonstrate that amebae migrating towards the "source" of a stable, spatial gradient move faster, extend fewer pseudopodia, and turn less frequently than amebae migrating away from the "source" in the same spatial gradient. In addition, amebae extend lateral pseudopods in a polarized fashion from the anterior half of the cell, and do so as frequently towards the source as away from the source. However, those formed towards the source more often produce a turn than those formed away from the source. These results suggest that there may be two decision-making systems, one localized in the pseudopods, and one along the entire cell body; they support the suggestion that Dictyostelium amebae may employ a temporal mechanism to assess the direction of a spatial gradient of chemoattractant.