Identification and Characterization of Force Sensitive Domains using an In Vivo Dorosphila Synthetic Biology Platform

Abstract

The Notch protein is a family of highly conserved transmembrane signaling proteins that are found in all metazoan life. This protein family plays many roles in developmental and regulatory pathways in these organisms such as the development of the human vertebral column and the Drosophila adult wings. The Drosophila Notch protein has been the focus of recent research as this protein has shown to be dependent on a unique signaling mechanism that relies on the force being applied to the receptor by the endocytosis of a bound ligand for a cleavage event to occur. This protein has been shown to only signal in the presence of this endocytosis force which allows for cleavage at the S2 and S3 sites on the protein which allows for a transcription factor to be released from the membrane and enter the nucleus. In synthetic biology, this protein has found a role in creating customizable cell-cell signaling platforms known as Synthetic Notch or Syn-Notch. As demonstrated in previous research, Notch proteins contain highly modifiable ligand binding and transcription factor domains that can be interchanged with other ligand binding and transcription factor domains from non-Notch proteins without affecting the protein signaling. In addition to the ligand-binding and transcription factor, the Notch protein contains a Negative Regulatory Region (NRR) which functions as a force sensor domain and allows for the force signaling activation seen in the native Notch proteins. This region of the Notch protein is often conserved in Syn-Notch systems as a means of controlling the signaling of Syn-Notch proteins used in precision medicine. The unique force-sensing function of this domain has raised questions regarding the characteristics of this domain that allow for it to act in a force-sensing manner and if this domain is as interchangeable as its other components. In this study, domains from other non-Drosophila Notch proteins and non-Notch protein domains were evaluated in an in vivo Drosophila platform for the ability to function as a force sensor domain. In this screen, several unique force sensor domains were identified as having the ability to recapitulate the force sensing characteristic of the canonical Notch NRR. While a common thread between these domains in terms of defining characteristics remains elusive, this study was able to demonstrate the feasibility of this screening method to identify force-sensitive domains and identify 4 domains that exhibit this characteristic. The identification of these domains which are force sensitive not only allows for the characteristics to function as force-sensitive domains but also provides alternative force sensors with differing force sensitivities when compared to the canonical Notch NRR for use in Syn-Notch systems. The development of these receptors will not only contribute to the field of synthetic biology as means to create synthetic platforms for use in future research, but these receptors will allow for the development of even more precise treatments using these synthetic receptor systems in clinical therapies.