Adenylyl cyclases and their product, 3'-5'-cyclic adenosine monophosphate (cAMP), are critical signaling molecules fundamental to many signaling pathways found throughout eukaryotes and mammals. This importance arises from the ability to not only transduce signals from receptors to downstream effectors but also to amplify the initial signaling response. In Dictyostelium, the binding of the chemoattractant cAMP to its G protein coupled receptor activates a variety of effectors including the adenylyl cyclase A (ACA). We demonstrate using the gene fusion product ACA-YFP (ACA and yellow fluorescent protein) that ACA is enriched at the back of chemotaxing cells and propose that this enrichment provides a compartment from which cAMP is released, relaying the chemotactic signal to neighboring cells and allowing the cells to align head-to-tail, forming streams during chemotaxis. Interestingly, we find that this enrichment of ACA is composed of many smaller vesicles containing ACA and that ACA containing vesicles are observed rapidly moving throughout the cell. We investigated the role of ACA vesicle trafficking in the enrichment of ACA at the back of cells. Using Fluorescence Recovery After Photobleaching (FRAP), we find that vesicle delivery of ACA-YFP to the plasma membrane is required for the asymmetric enrichment of ACA at the back of cells. When actin fibers and microtubules are disrupted with latrunculin A or nocodazole, respectively, ACA vesicle trafficking is strongly inhibited resulting in the loss of ACA enrichment at the back of cells. We find that ACA vesicles co-localize with microtubules and that nocodazole-treated cells have streaming defects. Together, these findings suggest that vesicle trafficking is required for cAMP release. Intriguingly, we also observe that migrating cells leave behind trails containing ACA. Since migrating cells maintain a polarized distribution of ACA, we reason that ACA must be replenished by protein synthesis to maintain its asymmetric distribution. To investigate this, we completely bleached migrating ACA-YFP expressing cells and monitored the fluorescence recovery over time. We observe a 40% recovery within 7 minutes, presumably due to protein synthesis. Indeed, we find that cycloheximide treatment reduces ACA levels, abolishes its enrichment specifically at the back of migrating cells and prevents streaming. Our findings provide a novel model to explain group cell migration, where vesicles containing de novo enzymes involved in the synthesis of chemoattractants are delivered to the back of migrating cells, thereby creating a compartment from which chemoattractants are specifically released. We propose that similar methods of enzyme compartmentalization exist in mammalian cells allowing for cells to migrate collectively to sites of inflammation, to metastasize to new tissues and to form neural networks.
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|Advisor:||Smith, L. Courtney, Parent, Carole A.|
|Commitee:||Brown, Ken M., Donaldson, Julie G., Kimmel, Alan R., Morris, David W.|
|School:||The George Washington University|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 69/11, Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Microbiology|
|Keywords:||Adenylyl cyclase, Chemoattractants, Dictyostelium, Migration, Streaming, Vesicle trafficking, cAMP|
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