The role of the I-BAR proteins MIM and IRSp53 in actin dynamics and development in Drosophila

UoM administered thesis: Phd

Abstract

The I-BAR proteins are a family of actin regulators which include IRSp53 and Missing-In-Metastasis (MIM). These proteins possess an N-terminal I-BAR domain which associates with both the actin cytoskeleton and membrane phospholipids and is able to induce membrane curvature. Previous cell culture and in vitro studies have implicated I-BAR proteins in the regulation of actin protrusion formation, however their roles within the organism are poorly understood. We have used Drosophila melanogaster as a model system in which to study I-BAR protein function at the cellular and organismal level. Drosophila possess two I-BAR proteins, one homologous to IRSp53 and one homologous to MIM. Using full- length and truncated splice variants generated for both dMIM and dIRSp53, we have performed structure/function analysis to determine the role of specific domains in the localisation and actin modifying abilities of the proteins in both cell culture and in vivo. We found that dMIM overexpression typically promotes a lamellipodial morphology, with dMIM localising to the edges of extending lamellipodia. dIRSp53 expression induced a more filopodial phenotype in cell culture, which was not as notable in vivo, however expression of a dIRSp53 splice variant with a WH2 domain resulted instead in a predominantly lamellipodial morphology. Similar to dMIM, dIRSp53 localises to the tip of extending protrusions, albeit more transiently. We found that multiple domains contribute to the localisation and activity of dIRSp53 and dMIM. Following overexpression analysis, complementary loss-of-function analysis was performed in vivo using Drosophila mutants lacking dMIM, dIRSp53 or both genes together. Surprisingly these mutants were viable and morphologically normal. Absence of these genes individually or together did not greatly affect cell migration or actin dynamics in haemocytes or epithelial cells undergoing dorsal closure. However, a role for I- BAR proteins in axonal filopodia formation within primary neuronal cultures was apparent, as was a notable role in neuromuscular junction morphology. We have also identified potential redundancy between Drosophila MIM and the Drosophila F-BAR protein Cip4 in actin bundle regulation within embryonic haemocytes, with an additional novel role for Cip4 alone in haemocyte lamellipodial maintenance. Our results suggest that the Drosophila I-BAR proteins contribute to actin cytoskeleton regulation in vitro and in vivo, particularly within the CNS, and with novel shared functions with other BAR domain family proteins contributing to their regulation of actin cytoskeletal organisation and function.

Details

Original languageEnglish
Awarding Institution
Supervisors/Advisors
Award date1 Aug 2013