The Activity of Coronin A is Dependent on a Protein(s) Secreted by Dictyostelium Discoideum in the Early Starvation Response

coronin A;conditioned medium;cell aggregation;cAMP relay;Biotechnology

Lubart, EmanuelCummings, Patrick ;

Johns Hopkins University

关键词: coronin A;    conditioned medium;    cell aggregation;    cAMP relay;    Biotechnology;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/38047/LUBART-THESIS-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

The social amoebae Dictyostelium discoideum is used widely as a model organism for studying development. It becomes chemotactic to autocrine signals following starvation, initiating a cell aggregation program in which different multicellular behaviors can be studied to elucidate mechanisms involved in development of eukaryotic cells. Starvation induces cell aggregation that plays an essential role in multicellular morphogenesis of the slime mold Dictyostelium discoideum. While secreted factors released by D. discoideum in the medium are known to regulate the developmental cycle in this amoebae have been identified, the downstream molecular signaling mechanisms involved in the transition from unicellularity to multicellularity remain largely uncharacterized. Our previous work suggests that cell aggregation is driven by coronin A that is required for D. discoideum to initiate the early development by activating the cAMP signaling pathway that in turn is essential for upregulation of genes involved in aggregation and development. In contrast to wild-type cells, cells lacking coronin A resulted in D. discoideum strains that undergo abnormal morphogenesis when incubated with factors from the starvation conditioned medium. These previous results suggest that the activity of coronin A is dependent on a specific factor(s) secreted by starving cells that regulate the transition from the nutrient deprivation to development stage. However, how coronin A regulates the organization of initial development, and which of these secreted factors is responsible for the activation of coronin A is not known. To characterize, purify, and identify the specific factor(s) required for coronin A-dependent development, heat-sensitivity, proteinase K, and phospholipase D treatments were performed. Our results implicate proteins as the biochemical entity inducing morphogenesis, which are heat-labile and protease-sensitive factors. Furthermore, ultrafiltration of conditioned medium suggested that the active fraction consists of a protein or protein complex with a molecular weight above 100.000 Dalton. We have further defined the properties of the active fraction as well as initiated purification of the protein(s). We have utilized ion-exchange chromatography, and were able to separate the activity into discrete fractions. In an attempt to screen candidates in these fractions for development-inducible factors, we compared cell aggregation activities in wild-type strains DH1-10 and coronin A-deficient Dictyostelium discoideum cells after further separation of the proteins by size exclusion chromatography. Our data suggested evidence for the requirement of a coronin A-dependent factor(s) in the cell aggregation process. In addition, our work revealed that ion exchange and gel filtration chromatography are suitable methods for characterizing the factors. Together, this study may contribute to a better understanding of the secreted factor(s), and their signaling pathways, and define the basis for regulatory mechanisms involved in the early starvation response of D. discoideum.