Vesicle trafficking and protein synthesis, target adenylyl cyclase A to the back of migrating cells localizing the release of chemoattractant to the trailing edge.

In Dictyostelium, the binding of the chemoattractant cAMP to its G protein coupled receptor activates a variety of effectors including the adenylyl cyclase ACA, which converts ATP to cAMP. We demonstrate 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 also found that ACA labels rapidly moving intracellular vesicles. 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 cannot stream. 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.

Author

• Kriebel, Paul

Language

• en

Keyword

• cAMP
• migration
• adenylyl cyclase
• chemoattractant
• Dictyostelium
• streaming

Date created

• 2009

Type of Work

• Dissertation

License

• All rights reserved

Rights statement

• In Copyright

GW Unit

• Biological Sciences

Degree

• Ph.D.

Advisor

• Smith, L Courtney
• Parent, Carole A

Committee Member(s)

• Brown, Ken M
• Kimmel, Alan R
• Morris, David W
• Donaldson, Julie G

Persistent URL

•  https://scholarspace.library.gwu.edu/etd/dj52w483c