A Novel Role for beta(Heavy)-Spectrin in Early Endosome Recycling in the Drosophila melanogaster Larval Midgut

Open Access
Author:
Phillips, Matthew D.
Graduate Program:
Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
June 06, 2005
Committee Members:
  • Graham Hugh Thomas, Committee Chair
  • Carol V Gay, Committee Member
  • Simon Gilroy, Committee Member
  • Richard W Ordway, Committee Member
Keywords:
  • Drosophila
  • midgut
  • cuprophilic
  • cell polarity
  • endocytosis
  • spectrin
Abstract:
Polarity is essential for the function of epithelial tissues. Epithelial polarity is marked by the division of the plasma membrane into apical and basolateral membrane domains, a division actively maintained by the delivery and retention of proteins in each domain. In parallel, an apicolateral junctional complex passively maintains the distinct biochemical composition of each domain by acting as a molecular fence. Both of these mechanisms for maintaining epithelial polarity depend to some degree on the cytoskeleton, a complex network of microtubules and F-Actin that is essential for polarized protein delivery, and interacts with integral membrane proteins and components of the junctional complex. The Spectrin-based membrane skeleton (SBMS) is a specialization of the actin cytoskeleton that forms a lattice on flat membranes. There it both regulates the transport of vesicles and provides a stable platform for the binding of adaptor proteins such as Ankyrin. The discovery that the Spectrin skeleton is polarized in epithelia, with different isoforms isolated to different domains, provides a mechanism to explain how differences in apical and basolateral domains are maintained. Spectrin tetramers are composed of two alpha and two beta subunits. beta-Spectrin isoforms are thought to be central for activities that contribute to the polarization of Spectrin tetramers, as well as their distinct functions. Drosophila melanogaster is ideal for studying the role of beta subunits in the function of the SBMS, since it only encodes two isoforms: a conventional beta and a beta heavy (betaH). The Drosophila Spectrin skeleton is polarized in epithelia, with basolateral (alpha/beta)2 and apical (alpha/betaH)2 tetramers. Previous results have shown that the Spectrin skeleton acts as a molecular scaffold, stabilizing a subset of membrane proteins at a particular membrane domain. However, my results point to a different effect on polarity. In betaH mutants, the apical Spectrin skeleton is lost, and defects in midgut acidification point to a loss of the apical proton-pumping V-type ATPase. Immunohistochemical work presented herein shows that the lack of midgut acidification is correlated with the loss of this V-ATPase not only from the apical membrane, but from a system of early endosomes involved in protein recycling. The early endosome pathway itself is compromised in these mutants, yet my work also shows that the delivery of apical proteins does not apparently require the SBMS. These results suggest that in betaH mutants an entire class of proteins normally recycled to and from the membrane may be prematurely degraded in the lysosome. For example, the trace mineral copper demonstrates a defective and lesser accumulation in betaH mutant midgut cells, suggesting the apically localized copper transporter Ctr-1 may also be affected. In addition, this work demonstrates functional differences in the polarizing activities of the epithelia derived from the surface ectoderm vs. the midgut. The C-terminal domain of betaH possesses a Pleckstrin Homology (PH) domain that interacts with membrane lipids and may be responsible for the delivery of the protein to the apical membrane. In the salivary gland, protein constructs made from the C-terminus of betaH do not show specific membrane localization, but instead cause the accumulation of membrane in large multifolded structures. My results in midgut, however, indicate that membrane delivery and retention at specific domains is contained in sequences C-terminal to the PH domain. Also, while I observe in the midgut a phenotype superficially similar to the membrane accumulations in salivary gland, the structures in midgut exhibit different positioning in the cell as well as a distinct protein composition. Finally, it is essential to study Spectrin function in clinically relevant systems. Spectrin mutations are associated with hemolytic anemias in humans, and neuronal pathologies in mice. A betaH isoform (beta5) has been reported in humans, where it preserves many features found in the fly and nematode proteins. Here I report and analyze the sequence of beta5 isoforms in mouse and chicken, which also contain many similarities to known b heavy isoforms. I also performed in situ hybridizations to determine the developmental expression profile of beta5 in mice, which show that it is either expressed at low levels or not at all through 10 days post conception. However, an anti-human beta5 antibody detects strong signal in adult mouse cardiac muscle and cerebellar axons of the white matter, suggesting that beta5 is essential for the function of these organs. The roles of unconventional beta-Spectrin subunits in cell polarity and animal development are just now being discovered. Therefore, the work presented in this thesis addresses both their structures and functions in both invertebrate and vertebrate model systems.