THE ROLE AND REGULATION OF APICAL SPECTRINS IN CELL POLARITY, TRAFFICKING, GROWTH, AND CORTICAL TENSION IN DROSOPHILA MELANOGASTER
Open Access
- Author:
- Browder, Kristen Cherie
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 15, 2016
- Committee Members:
- Graham H. Thomas, Dissertation Advisor/Co-Advisor
Graham H. Thomas, Committee Chair/Co-Chair
Melissa Rolls, Committee Member
Lorraine Santy, Committee Member
William Hancock, Outside Member
Lu Bai, Committee Member - Keywords:
- Crumbs
Protein Phosphatase 2A (PP2A)
Tension
Beta Heavy-Spectrin
Alpha Spectrin
Polarity
Par6
Sdt
aPKC
Strain Sensor
Endocytosis
Hippo/Warts Pathway
cpstFRET
Drosophila - Abstract:
- Spectrins are long, rope-like proteins that form heterotetramers of two and two chains. These heterotetramers crosslink F-actin in the cell to form a structural network call the Spectrin-Based Membrane Skeleton (SBMS). In Drosophila melanogaster, there are three genes that encode spectrins: alpha-spec (encoding alpha-spectrin), β-spec (encoding conventional β-spectrin), and karst (encoding βH‑spectrin; βH) (Moorthy et al., 2000; Thomas and Williams, 1999; Bennett and Baines, 2001; Bennett and Healy; 2008). The SBMS is not only important for maintaining cell structure, but also plays roles in cell polarity, membrane trafficking, cell growth, and cortical tension. This dissertation reports two major studies involving the role of spectrin in polarity, trafficking, and growth (Chapter 2) and cortical tension (Chapter 3). βH is apically localized in epithelial cells and interacts with many protein partners that contribute to its diverse functions (outlined in Chapter 1). One of these interactions is with the apical determinant Crumbs (Crb) to regulate apical membrane size. βH is also required for correct endosomal trafficking to and at the multivesicular body (MVB) and in recycling of proteins to the plasma membrane. Using a yeast two-hybrid screen, a subunit of Protein Phosphatase 2A (PP2A) was shown to directly interact with βH. PP2A is a family of serine/threonine phosphatases involved in many important cellular events. This heterotrimeric protein is comprised of a catalytic (C) subunit and structural (A) subunit. The third, variable regulatory B subunit, determines the substrate specificity, localization, and catalytic activity of the PP2A enzyme. Chapter 2 presents data demonstrating that PP2A-PR72 (a regulatory B subunit) is involved in modulating both the βH/Crb complex during apical pole establishment/maintenance, and endomembrane trafficking. PP2A-PR72 knockdown flies exhibit an elevated number of late endosomal compartments when stained for the late endosome marker Rab7 and an accumulation of acidic compartments when stained with LysoSensor. In addition, βH becomes internalized and localizes to Hrs positive MVB. This suggests that PP2A-PR72 normally down regulates lysosomal trafficking, encouraging protein recycling as previously hypothesized for the action of βH and its molecular partner Annexin B9 (Tjota et al., 2009). An extensive series of genetic interaction experiments using various Crb, aPKC, and Hippo pathway constructs in conjunction with PP2A-PR72 knockdown and overexpression, and immunostaining in larval salivary glands, suggests that PP2A-PR72 negatively regulates Crb activity, specifically in its regulation of the Hippo/Warts pathway via Expanded. When overexpressing PP2A-PR72 in adult wings there is a significant size increase compared to wild-type or PP2A-PR72 knockdown consistent with Hippo downregulation. Also, genetic interaction experiments with Yorkie/PP2A-PR72 co-overexpression suggest that PP2A-PR72 negatively regulates the Hippo/Warts pathway. The interaction seen is strikingly similar to the PP2A-PR72 interaction with Crb, suggesting that PP2A-PR72 modulates Crb well documented cross-regulation of the Hippo/Warts pathway. Beyond spectrin’s roles in polarity, trafficking, and growth, the SBMS is constantly adapting to changes in cell shape during epithelial morphogenesis. It does so, in part, by reversible folding and unfolding of spectrin repeats. The unfolding force required for repeat unfolding is low at a range of 25-35 pN. A FRET based strain sensor inserted within an alpha-spectrin repeat was used to observe and measure tension in the spectrin network in the terminal web of the gut epithelia and in apically contracting cells during embryogenesis (Chapter 3). This strain sensor confirmed that spectrin is experiencing tension in actively contracting cells. Upon ablation this tension is relieved in step with recoil of the tissue, and spectrin network tension returns during wound repair, specifically as it accumulates around the leading edge of the wound. The actomoyosin network also accumulates at this site suggesting a collaboration with the SBMS. Myosin-II and alpha-spectrin do co-localize within cells known to be actively contracting during embryogenesis supporting this collaboration. Live imaging experiments are on-going to confirm the connection between the SBMS and actomyosin network. Chapter 4 contains a comprehensive model extending known information in the literature in Chapter 1 with data presented in Chapters 2 and 3 of βH roles in polarity, trafficking, growth, and cortical tension as it relates to Crb trafficking. A step-by-step approach is used to explain the order of events as well as several hypotheses for PP2A-PR72 regulation in early stages of endocytosis and growth.