Graduation Term

2020

Degree Name

Master of Science (MS)

Department

School of Biological Sciences

Committee Chair

Kevin Edwards

Abstract

Mutations in the giant actin-membrane linker protein Dystrophin (Dys) are the cause of Duchenne Muscular Dystrophy (DMD). In the Drosophila model system, the highly conserved Dys protein is not required for viability but is required for proper wing and ovarian development. Dys mutations produce two visible phenotypes: posterior crossveins are detached from the longitudinal veins, and in oogenesis the developing eggs fail to elongate properly. This provides an opportunity to explore the less-understood cellular and developmental roles of Dys and gain a new insight into how tissues adopt their correct shapes.

Animals from worms to flies to humans express multiple Dys isoforms that vary in length; the long forms contain the actin-binding domains (ABD) connecting the cortical F-actin to the extracellular matrix (ECM). The short forms however, lack the ABD and should not be able to act as membrane-actin linkers, but could scaffold and regulate signaling pathways associated with Dys. Probing the functional differences of these isoforms is important not only for basic cell and developmental biology but also for understanding DMD and treating it with truncated or partially restored proteins. We characterized a transposon insert in the Dys coding region which only removes the long isoforms. Remarkably, it behaves as wild type for the egg elongation phenotype but is null for the wing phenotype. A Dys allele with a GFP exon, which labels the long forms but not the shortest class of isoforms, shows Dys is organized in a plane polarized striated pattern, and associates with F-actin in a dynamic, stage-dependent manner. A Dys monoclonal antibody labels all the isoforms and detects a similar but extended striated pattern of Dys. Since each tag labels no more than one point along the protein, the pattern implies multiple copies of Dys are aligned, and two or more isoforms are coordinated in these structures.

Dys null mutants lose F-actin plane polarization in the basal follicle cell epithelium, and gain excessive cell surface projections. Germline and muscle RNAi against Dys do not trigger the typical Dys phenotypes. However, follicle cell-specific depletion of Dys results in short eggs and disorganized actin at the egg's exterior indicating Dys is required in the follicle cells for proper morphogenesis.

Dys and another structurally related member of the spectrin-repeat superfamily called beta-Heavy spectrin (βH-Spec), overlap in their developmental functions. More specifically, βH-spec;Dys double mutants show high lethality, but also fused cysts, demonstrating a novel functional network at the cell cortex involving the spectrins and Dys. Together, these results highlight previously unknown aspects of functional and subcellular organization for Dys which is critical for our understanding of development and for regenerative medicine.

Access Type

Thesis-Open Access

DOI

https://doi.org/10.30707/ETD2020.1604319249173

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