Universität zu Köln

Kroll, Patrizia ORCID: 0000-0002-7308-2292 (2024). Analysis of Smallish function in the regulation of actomyosin contractility during epithelial morphogenesis in Drosophila melanogaster. PhD thesis, Universität zu Köln.

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Abstract

The cortical actomyosin cytoskeleton generates the mechanical force driving highly dynamic processes occurring during epithelial morphogenesis, such as cell shape changes during cell division, cell intercalation, and the formation of furrows and tubes. The LIM domain protein Smallish (Smash) was shown to modify actomyosin contractility during morphogenesis since smashΔ35m/z null mutants show reduced membrane tension, whereas overexpression of Smash leads to apical constriction in the follicle epithelium. Many smashΔ35m/z null mutant embryos show strong epithelial morphogenetic defects, likely due to misregulation of the actomyosin cytoskeleton. Although we have several lines of evidence demonstrating that Smash is functioning in a large protein interaction network to control membrane tension via the actomyosin cytoskeleton, the precise mechanism of how Smash functions remains to be elucidated. This study provides new insight into the molecular working mechanism of Smash. We showed that Smash is required for efficient phosphorylation of the non-muscle myosin II regulatory light chain encoded by spaghetti squash (sqh). The effect of Smash on Sqh phosphorylation appeared to be indirect because Smash did not influence Rok kinase activity in vitro. Instead, a novel dominant negative allele of smash provides evidence that Smash might be involved in regulating signaling upstream of Rok. In addition, we demonstrated that the myosin binding domain of Smash is crucial to induce actomyosin contractility. We suggest that the myosin binding domain of Smash is necessary to provide scaffolding function for Rok and Myosin II, thereby enabling Sqh phosphorylation, which in turn activates actomyosin contraction driving cell shape changes. A detailed investigation of the smash null and dominant negative allele demonstrated the importance of Smash in regulating the actomyosin cytoskeleton during epithelial morphogenesis, especially during germ band elongation. The generation of CRISPR/Cas9 GFP knock-in lines expands our molecular toolkit to study the spatiotemporal behavior of Smash during morphogenesis, since the sfGFP knock-in line of SmashPM faithfully reflected the expression pattern and subcellular localization of Smash as described using an N-terminal α-Smash antibody. Moreover, the novel sfGFP knock-in lines revealed isoform-specific expression of SmashPI in posterior follicle cells during late oogenesis. Our phenotypic data were supported by the results obtained by a biotin proximity labeling assay, which was used to identify novel Smash-interacting proteins. This assay identified several promising actomyosin-associated proteins as potential Smash interaction partners. Among those were some with a reported function in embryonic morphogenesis and some others uncover a potential involvement of Smash in establishing oocyte polarity, ovulation, muscle development, cytokinesis, or regulating gene expression. A detailed analysis of these proteins and their interaction with Smash during morphogenesis and the other processes is likely to reveal important new insight into the mechanisms of actomyosin regulation.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Kroll, Patrizia patrizia.kroll@outlook.de orcid.org/0000-0002-7308-2292 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-742976
Date:	2024
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Medicine > Anatomie > Institut I für Anatomie
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language Smallish/dLMO7 English Actomyosin English Planar cell polarity English Morphogenesis English Rho kinase English
Date of oral exam:	17 January 2024
Referee:	Name Academic Title Wodarz, Andreas Prof. Dr. rer. nat. Roth, Siegfried Prof. Dr. rer. nat.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/74297