Investigating the role of βheavy-spectrin in modulation of Rac1 activity

Open Access
Crilly, Stephanie E
Area of Honors:
Biochemistry and Molecular Biology
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Graham Hugh Thomas, Thesis Supervisor
  • James Endres Howell, Honors Advisor
  • spectrin
  • rac
  • epithelial polarity
  • trafficking
Previous research supports a model for Drosophila melanogaster in which the C-terminal segment of βHeavy-spectrin (βH33) and the Rac1 GTPase have an antagonistic relationship, which appears to be important for apical domain maintenance and formation of the zonula adherens. It is well known that the Rac1 effector Pak down-regulates βH at the apical domain. However, the mechanism and potential negative regulator of Rac1 signaling associated with βH33 is unknown. βH33 contains multiple interaction domains, which could facilitate the recruitment of a negative regulator of Rac1. Elucidation of the region of βH33 responsible for the observed genetic interaction, through further genetic interaction studies, provides insight into which interaction partners of βH33 play a role in the negative regulation of Rac1 activity. The residues to the C-terminal side of βH33 pleckstrin homology (PH) domain appear to be important for the observed genetic interaction. This region of βH33 includes the binding sites for the direct interaction partners of βH, PP2A regulatory subunit PR72 and AnxB9. Additionally, the apparent association of the Rac1 GEF Trio with an endosomal compartment positive for Hrs, a marker for the multivesicular body, along with the involvement of βH in endosomal trafficking, suggests that βH33-mediated Rac1 downregulation could be occurring at the endosomal level. Immunofluorescence characterization of Trio localization in eye and wing imaginal discs under wild-type conditions, suggests that Trio may be present on some subset of endosomal compartments and somewhat enriched on the early multivesicular body. Similar characterization under various knockdown and overexpression conditions, indicates that βH33-mediated modulation of Rac1 activity may not be occurring through direct regulation of Trio protein levels or distribution in the cell, though further studies are needed.