Poster abstracts
Poster number 110 submitted by Adalyn Brown
A single molecule approach to detect the oligomeric changes associated with EphA3 activation in live cells
Adalyn Brown (Molecular Cellular and Developmental Biology), Jacob Smith (Ohio State Biochemistry Program), Vladislav Belyy (Chemistry and Biochemistry Department)
Abstract:
Eph receptors are a family of receptor tyrosine kinases that regulate cellular communication, tissue organization, and cell migration, making them critical in development and disease. Dysregulation of Eph signaling has been implicated in cancer and neurodegenerative disorders. While ephrin-induced receptor clustering is required for activation, how large these oligomers get and how oligomer stoichiometry affects downstream signaling is still not fully understood. Expression of individual Eph receptors varies among cell lines and throughout development, and the relationship between expression level and activity of a specific receptor remains uncharacterized. In this study, we investigate the ephrin-dependent signaling of the low-abundance EphA3 receptor in prostate cancer cell lines. We confirmed the minimal expression of EphA3 in the PC-3 cell line by transcriptome sequencing and immunoprecipitation. We developed an EphA3-HaloTag construct that can be used for single-molecule tracking experiments and confirmed that the HaloTag fusion does not prevent the chimeric protein from responding to ephrin. We are currently carrying out live-cell imaging experiments to find the baseline oligomeric state of EphA3-HaloTag in PC3 cells and probe how its oligomerization changes upon stimulation. We also plan to examine heterodimerization between EphA3 and the highly expressed receptor EphA2 by comparing oligomer sizes in EphA2 knockout and wild-type PC-3 cells expressing EphA3, providing further insight into the role heterooligomers may play in influencing oligomerization and cell signaling. We will assess transcriptomic differences between activated and non-activated states using Nanopore sequencing to gain a better understanding of the transcriptomic changes resulting from Eph receptor activation. Future work will also involve CRISPR-mediated manipulation of EphA3 in cell lines with endogenous expression to further define its role in receptor organization and signaling.
Keywords: oligomerization, microscopy, Receptors