BIOL, BMMB, MCIBS
Research in my lab asks fundamental questions about the roles of the cytoskeleton at the cell membrane in epithelial cells, including issues of cell polarity and adhesion, cell signaling, and morphogenesis. Drosophila is our chosen model system because of the multidisciplinary combination of tools available, and because of its well-characterized development. We use both molecular and cellular techniques as well as classical and transgenic genetic approaches.
The spectrin-based membrane skeleton is a ubiquitous structure that is conserved in diverse organisms. Spectrins are long heterotetramers of two α and two β chains, which crosslink F-actin and contain numerous protein binding sites along their length. Two dimensional spectrin networks form in association with various cell membranes where they can modulate cell shape, cell integrity and protein trafficking. Typically different spectrins are polarized to distinct parts membrane domains and are thus believed to contribute to cell polarity. Drosophila provides a simple model system for examining this molecular scaffold, since the fly has only one α and two β-genes: the type of spectrin thus depends on which β chain is used. Our goal is to understand how differentiation in the membrane skeleton is used to polarize cells in a developmental context.
We are currently focusing on one of the β-spectrin isoforms, βHeavy-spectrin (βH), which is associated with the zonula adherens, apical microvillar fields and morphogenetic movements driven by cytoplasmic myosin II. The distribution of βH during early embryogenesis suggests a role in early events during the development of apicobasal polarity, and mutations in the karst locus encoding this protein cause a number of defects in tissues of epithelial origin. Phenotypic analysis of karst mutants reveals that βH is necessary for maintaining the integrity of the zonula adherens, but is not required for apicobasal polarization per se. However, molecular and genetic analysis has also shown that βH is associated with the apical polarity determinant Crumbs which recruits βH to the membrane via a domain that has been shown to be important for zonula adherens development. Further characterization of the karst phenotype as well as gain-of-function phenotypes caused by βH sub-domain overexpression has revealed that βH has a role in maintaining protein levels at the plasma membrane through a novel role in protein recycling.
We also maintain an interest in the evolutionary origins of the membrane skeleton and have previously collaborated with Dr. Andrew Clark (Cornell) and Dr. Spencer Muse (North Carolina State University) to generate a comprehensive model for the evolution of the α-actinin/spectrin/dystrophin superfamily of proteins. We have found evidence that the ancestral gene structures of this superfamily were unstable during an early phase in their evolution and that this phase was dominated by concerted evolution. This has been followed by long-term stability in gene organization and a lack of sequence exchange between them. This model has general applicability for other proteins with repetitive structures.
My laboratory provides training in a variety of techniques that have wide applicability to other experimental systems. Furthermore, our multidisciplinary approach means that a typical experiment might include several of these. Experiments currently in progress use standard molecular biological techniques (such as PCR, cloning, sequencing and bacterial protein expression), membrane purification and proteomics, the generation of transgenic flies expressing mutant proteins, immunofluorescent microscopy with digital image acquisition and the analysis of genetic interactions.
The development of the wild-type Drosophila eye during early-mid pupation. Insect eyes are incredibly regular structures. The outline of each cell is labeled with α-catenin-GFP at cell-cell junctions. The cells can be seen to reorganize themselves into a near crystalline array by 28 hours after pupariation. This junctions is disrupted in the absence of spectrin function. (see J. Cell Sci. 123;277)
Combining genetics and biochemistry: Proteomic analysis of cell surface proteins from fly brains. We believe that the amount of a protein at the cell surface will be strongly influenced by spectrin function. In order to test this hypothesis we will compare the amount of every surface protein between wild-type and spectrin mutant tissues. This image shows our part of our membrane purification strategy (top panel -a density gradient), gel analysis of the final proteome (A) and proteomic analysis of proteins identified by mass spectrometry (B,C). Only low levels of non-plasma membrane compartments remain and the proteins identified in the definitive plasma membrane fraction have appropriate functions to be at the cell surface (C).
Funding in my lab comes from the National Science Foundation.