Adenylyl Cyclase Localization Regulates Streaming during Chemotaxis

Abstract

We studied the role of the adenylyl cyclase ACA in Dictyostelium discoideum chemotaxis and streaming. In this process, cells orient themselves in a head to tail fashion as they are migrating to form aggregates. We show that cells lacking ACA are capable of moving up a chemoattractant gradient, but are unable to stream. Imaging of ACA-YFP reveals plasma membrane labeling highly enriched at the uropod of polarized cells. This localization requires the actin cytoskeleton but is independent of the regulator CRAC and the effector PKA. A constitutively active mutant of ACA shows dramatically reduced uropod enrichment and has severe streaming defects. We propose that the asymmetric distribution of ACA provides a compartment from which cAMP is secreted to locally act as a chemoattractant, thereby providing a unique mechanism to amplify chemical gradients. This could represent a general mechanism that cells use to amplify chemotactic responses.

The Group Migration of Dictyostelium Cells Is Regulated by Extracellular Chemoattractant Degradation

“…Although both models are capable of reproducing many features of Dictyostelium aggregation, including the propagation of cAMP waves in characteristic spirals and the formation of stream-like aggregates, they are not perfect. In previous work, we have proposed that the secretion of cAMP is spatially restricted (Kriebel et al, 2003(Kriebel et al, , 2008. We now show that only the secreted form of PdsA can degrade the external cAMP signal and that the addition of a uniform bolus of PdsA does not lead to the formation of highly branched, head-to-tail lines characteristic of WT streaming.…”

“…Interestingly, as cells form aggregates, they acquire the ability to migrate in groups by aligning in a head-to-tail manner, forming characteristic streams. We have previously shown that the ability of cells to stream depends on the enrichment of ACA-containing vesicles at the back of migrating cells (Kriebel et al, 2003(Kriebel et al, , 2008. We propose that the formation of head-to-tail arrays is regulated by the specific release of cAMP from vesicles at the back of cells.…”

“…In all experiments, UK5 and UK7 cells behaved similarly; for consistency, findings obtained with UK5 cells (which we refer to as pdsA Ϫ ) are presented in this study. WT (AX3) pdsA Ϫ or transfected cells were grown in shaking cultures in HL5 media to ϳ5 ϫ 10 6 cells/ml and processed for development as described previously (Kriebel et al, 2003). Briefly, cells were washed once in development buffer (DB; 5 mM Na 2 HPO 4 , 5 mM NaH 2 PO 4 , pH 6.2, 2 mM MgSO 4 , and 200 M CaCl 2 ), resuspended in DB at 2 ϫ 10 7 cells/ml, and shaken at 100 rpm for 4 -8 h with pulses of 75 nM cAMP every 6 min.…”

“…Because the enrichment of ACA at the back of migrating cells is also required for cells to migrate in a head-to-tail manner, we transfected pdsA Ϫ cells with our ACA-YFP expression plasmid (Kriebel et al, 2003) and assessed the cellular distribution of ACA during chemotaxis and streaming. To further characterize the behavior of pdsA Ϫ /PEC cells, we also monitored the distribution of the cAMP receptor cAR1-GFP and CRAC-GFP, a pleckstrin homology domain-containing protein necessary for ACA activation and development, in these cells during chemotaxis.…”

Oscillatory signaling and network responses during the development of Dictyostelium discoideum

“…Significantly, ACA-YFP fusion proteins localize to intracellular vesicles and to the rear of polarized chemotaxing cells (Kriebel et al, 2003;Figure 3). In a mixed population of aggregating WT and aca-null cells, the placement of an aca-null within a stream terminates signal propagation.…”