Biochemical Analysis of Slam – a polarity protein during early Drosophila embryogenesis

by Stephanie Häusser née Gröning
Doctoral thesis
Date of Examination:2021-09-07
Date of issue:2021-09-23
Advisor:Prof. Dr. Jörg Großhans
Referee:Prof. Dr. Jörg Großhans
Referee:Prof. Dr. Heike Krebber
Persistent Address: http://dx.doi.org/10.53846/goediss-8832

 

 

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Abstract

English

Slow as molasses (slam) is a gene essential for embryonic development of Drosophila. Slam is necessary for the formation and ingression of the plasma membrane and its polarisation, i.e. the formation of the basal cortical domain. Later, Slam controls the directed migration of primordial germ cells. Slam may organise a platform for Rho signalling and membrane trafficking. Slam displays the striking feature that Slam protein and slam mRNA colocalize at the basal domain and form a complex which can be isolated by immunoprecipitation. The interaction of the protein and the mRNA at the basal domain of the plasma membrane is important for the mRNA localization and its efficient translation. However, little is known about the molecular mechanisms by which Slam fulfils its tasks. Slam encodes an unconserved protein of 1196 amino acid residues with no obvious domains and with large regions predicted to be structurally disordered. This study aims to further investigate the biochemistry and molecular interactors of Slam to advance the understanding of membrane formation and polarisation. Firstly, a series of Slam truncations were constructed and expressed in E. coli to identify Slam fragments which may be suitable for crystallographic studies in the future. Investigating Slam’s structure will give fundamental insights into its nature and way of function. A protocol for the purification of the C-terminal 129 aa region of Slam was developed and optimised to generate high-purity submilligram amounts of the fragment. Secondly, the proteome of Slam interactors was isolated and identified by mass spectrometry. To this end, a multistep immunoprecipitation protocol was developed. Previously known interactors were confirmed. Strikingly, the interactors fall into functional classes. Multiple components of the actin cytoskeleton were isolated, such as components of the Arp2/3 complex, alpha-actinin and non-muscle Myosin II. Furthermore, components related to the cell cortex and membrane trafficking were found, including Cindr, Restin homolog (clip190) and Jaguar (Myosin 95F). The list of interactors will serve as a starting point for future studies resolving the role of Slam for membrane formation and its interaction with the actin cortex.
Keywords: Slam; Actin; Cellularization; Drosophila; Drosophila embryogenesis; actin binders; adaptor protein
 
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