BETA H-SPECTRIN--ROLES IN CELL POLARITY AND SIGNALING

Project: Research project

Project Details

Description

The long term goal of this study is to understand the composition and
function of the spectrin-based membrane skeleton in non-erythroid
cells. The membrane skeleton was discovered in erythrocytes and is now
very well defined in that cell type. Isoforms of many of the
erythroid membrane skeleton components exists in many if not most non-
erythroid cell types. Although it has been postulated that regional
variation in the membrane skeleton may participate in the process of
generating cell polarity in non-erythroid tissues, the specific roles
of the various spectrin isoforms with distinct tissue and subcellular
localization's in non-erythroid cells is still unclear.

We have chosen Drosophila as a model system since it provides a unique
and powerful combination of genetic, molecular and cellular tools with
which to achieve our goals. Specifically, this proposal will study
beta/heavy-spectrin (betaH), an isoform of beta-spectrin in this
organism. We have created mutations in the betaH gene, karst, and
find that these are lethal. This indicates that betaH is crucial for
the development of the fly, and provides a unique opportunity to study
the role of a specific beta-spectrin isoform in vivo. Preliminary
analysis of the karst phenotype has revealed defects in tissues of
epithelial origin. This application focuses on one of these defects:
failure of the receptor tyrosine kinase/ras-based sevenless signaling
pathway that normally determines a specific cell fate in the
developing eye imaginal disc. Based on our preliminary data, we
hypothesize that betaH is involved either in the apical restriction
of sevenless signaling components, in cell adhesion between the
communicating cells or in building microvillar structures containing
signaling components. We will take a number of complementary
approaches to identify the most likely of these hypotheses.

Our specific aims are: i) to make a rescue construct suitable for
directed mutagenesis of the betaH sequence, ii) to characterize
defects in epithelial cell architecture in karst mutant eyes by
staining with a battery of antibodies to key proteins, iii) to
identify specific domains of betaH that play a role in sevenless
signaling, iv) to identify interacting proteins by characterizing
complexes containing betaH that we will identify by
immunoprecipitation.

The proposed experiments will make a fundamental contribution to our
understanding of epithelial structure: one the first lines of defense
against most human pathogens. They will also illuminate the
relationship between ras signaling and the cytoskeleton, a
relationship that is important for oncogenic and metastatic processes.
StatusFinished
Effective start/end date5/1/974/30/98

Funding

  • National Institute of General Medical Sciences

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