Delineating the core regulatory elements crucial for directed cell migration by examining folic-acid-mediated responses.

Dictyostelium discoideum shows chemotaxis towards folic acid (FA) throughout vegetative growth, and towards cAMP during development. We determined the spatiotemporal localization of cytoskeletal and signaling molecules and investigated the FA-mediated responses in a number of signaling mutants to further our understanding of the core regulatory elements that are crucial for cell migration. Proteins enriched in the pseudopods during chemotaxis also relocalize transiently to the plasma membrane during uniform FA stimulation. In contrast, proteins that are absent from the pseudopods during migration redistribute transiently from the PM to the cytosol when cells are globally stimulated with FA. These chemotactic responses to FA were also examined in cells lacking the GTPases Ras C and G. Although Ras and phosphoinositide 3-kinase activity were significantly decreased in Ras G and Ras C/G nulls, these mutants still migrated towards FA, indicating that other pathways must support FA-mediated chemotaxis. We also examined the spatial movements of PTEN in response to uniform FA and cAMP stimulation in phospholipase C (PLC) null cells. The lack of PLC strongly influences the localization of PTEN in response to FA, but not cAMP. In addition, we compared the gradient-sensing behavior of polarized cells migrating towards cAMP to that of unpolarized cells migrating towards FA. The majority of polarized cells make U-turns when the cAMP gradient is switched from the front of the cell to the rear. Conversely, unpolarized cells immediately extend pseudopods towards the new FA source. We also observed that plasma membrane phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] levels oscillate in unpolarized cells treated with Latrunculin-A, whereas polarized cells had stable plasma membrane PtdIns(3,4,5)P3 responses toward the chemoattractant gradient source. Results were similar for cells that were starved for 4 hours, with a mixture of polarized and unpolarized cells responding to cAMP. Taken together, these findings suggest that similar components control gradient sensing during FA- and cAMP-mediated motility, but the response of polarized cells is more stable, which ultimately helps maintain their directionality.

Determination of postexcitation thresholds for single ultrasound contrast agent microbubbles using double passive cavitation detection.

This work presents experimental responses of single ultrasound contrast agents to short, large amplitude pulses, characterized using double passive cavitation detection. In this technique, two matched, focused receive transducers were aligned orthogonally to capture the acoustic response of a microbubble from within the overlapping confocal region. The microbubbles were categorized according to a classification scheme based on the presence or absence of postexcitation signals, which are secondary broadband spikes following the principle oscillatory response of the ultrasound contrast agent and are indicative of the transient collapse of the microbubble. Experiments were conducted varying insonifying frequencies (0.9, 2.8, 4.6, and 7.1 MHz) and peak rarefactional pressures (200 kPa to 6.2 MPa) for two types of contrast agents (Definity and Optison). Results were fit using logistic regression analysis to define pressure thresholds where at least 5% and 50% of the microbubble populations collapsed for each frequency. These thresholds were found to occur at lower pressures for Definity than for Optison over the range of frequencies studied; additionally, the thresholds occurred at lower pressures with lower frequencies for both microbubble types in most cases, though this trend did not follow a mechanical index scaling.