| Cell polarity and junctional integrity are key mechanisms for organism survival. The asymmetric distribution of proteins, along the plasma membrane and within the cells, is vital to proper cellular function as mis-localization or lack of these molecules often leads to diseases and sometimes death. Proteins such as Bazooka, Crumbs, Cadherins, Catenins, Scribble, and Lethal giant larvae are but a few conserved molecules (with homologues found in many species) that have been shown to play important roles in generating cell polarity. The role of the actin and spectrin cytoskeleton underlying these important protein complexes has yet to be elucidated. Thus, my investigations focused on determining the function of the spectrin based membrane skeleton (SBMS).;My research has focused on the role of the SBMS in cell polarity and the maintenance of junctional integrity, and specifically on how the beta Heavy-spectrin protein contributes to these processes, using Drosophila melanogaster as my model organism to study. The asymmetrically localized Drosophila spectrins: alpha, beta, and beta Heavy-spectrin (betaH) form (alphabeta)2 and (alphabetaH)2 heterotetramers that are restricted to the basolateral and apical domains in epithelia, respectively. These proteins are not unique to Drosophila; homologues have been found in vertebrates and invertebrates and are thought to act as molecular scaffolds since they contain various motifs that facilitate protein-protein interaction and membrane association. Data from several other labs have shown that these molecules are essential.;This dissertation details my investigation of betaH's role during Drosophila melanogaster development focusing on: (i) how and where betaH interacts with the apical determinant Crumbs; (ii) the dependence of betaH on DMoesin for membrane association; (iii) whether the pleckstrin homology (PH) containing C-terminal domain of betaH is responsible for membrane association and (iv) how betaH might be associated with Drosophila melanogaster protein Annexin B9 (Anx B9). The first, and collaborative, investigation shows that in Drosophila Schneider 2 (S2-embryonic cell line), transfected with the cytoplasmic tail of Crumbs, betaH interacts with the FERM binding domain of Crumbs. Genetic studies of the interaction between these two proteins show that removing crumbs dominantly enhances the lethality seen in the absence of karst (gene encoding betaH). Mosaic clonal analysis was conducted to address the second component, where DMoesin ovary clones show that in the absence of DMoesin, beta H was still associated with the apical plasma membrane, suggesting that beta H does not depend on DMoesin for its membrane association. Third, using C-terminally derived deletion constructs, my studies show that the pleckstrin homology (PH) domain is not solely responsible for beta H membrane association. Over-expression of these constructs caused increased membrane area and bi-membrane structures that were extensively colocalized with the endocytic protein Dynamin, suggesting that betaH might be involved in regulating membrane turnover. The final aspect of my investigation was concentrated on the possible association between betaH and Annexin B9 (Anx B9). Thus, I generated an antibody to Drosophila Annexin and for the first time show that Drosophila Anx B9 is localized to the cytoplasm and on the plasma membrane. My results also show that Anx B9 co-localizes with the bi-membrane structures generated by the over-expression of the C-terminal domain of betaH. Anx B9 loss-of-function analysis (RNAi) studies show that a decrease in the amount of Anx B9 causes a reduction of membrane-associated C-terminus and leads to a decrease in the number of bi-membrane structures, suggesting that Anx B9 is at least partly responsible for tethering betaH to the plasma membrane.;The culmination of this work shows that, while the underlying spectrin based membrane skeleton (betaH in particular) may not be essential for generating cell polarity, it does play an important role in regulating membrane turnover by interacting with molecules such as Crumbs, Dynamin and Annexin B9. |
