Drees, L. | bioRxiv Science

bioRxiv2023

The proteolysis of ZP proteins is essential to control cell membrane structure and integrity of developing tubes

Membrane expansion integrates multiple forces to mediate precise tube growth and network formation. Defects lead to deformations, as found in diseases such as polycystic kidney diseases, aortic aneurysms, stenosis, and tortuosity. We identified a mechanism of sensing and responding to the membrane expansion of tracheal tubes. We show in Drosophila that Zona Pellucida domain proteins Piopio and Dumpy cooperate to integrate mechanical stress at cell membranes and luminal matrix. When tension appears at the apical membrane due to tracheal tube length expansion, Piopio undergoes ectodomain shedding by the Matriptase homolog Notopleural, which releases Piopio-mediated linkages between membranes and extracellular matrix. Failure of this process leads to deformations of the apical membrane and comprises tubular network function. We also show conserved ectodomain shedding by the human matriptase during TGF-{beta} signaling, both of which are required in the lung, providing novel approaches for in-depth analysis of pulmonary diseases caused by cell and tube shape changes.

developmental biology↗

bioRxiv2021

Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence background of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating measurement of expression kinetics both in isolated cells and in whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a strong foundation for the development and expansion of transgenic techniques in this unique model system. MotivationThe study of planarians has contributed to advances in our understanding of regeneration, stem cell dynamics, and many other fundamental biological processes. However, the persistent challenge of expressing transgenes in planarians has led to the speculation that they may be resistant to transfection. In this work, we develop methods to express exogenous mRNAs in both isolated planarian cells and whole animals by optimizing delivery techniques, genetic constructs, and detection methods. These methods allow us to study transfection kinetics and post-transcriptional regulation of gene expression in a quantitative manner. Beyond planarian research, this work should also provide a broadly applicable strategy to develop similar tools for animals that are also challenging to modify genetically.

bioengineering↗