Identification and Characterization of Force Sensitive Domains using an In Vivo Dorosphila Synthetic Biology Platform
AbstractThe 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.
Showing items related by title, author, creator and subject.
Ndel1 Promotes Axon Regeneration via Intermediate FilamentsToth, Cory; Shim, Su Yeon; Wang, Jian; Jiang, Yulan; Neumayer, Gernot; Belzil, Camille; Liu, Wei-Qiao; Martinez, Jose; Zochodne, Douglas; Nguyen, Minh Dang; et al. (2008-04-23)Failure of axons to regenerate following acute or chronic neuronal injury is attributed to both the inhibitory glial environment and deficient intrinsic ability to re-grow. However, the underlying mechanisms of the latter remain unclear. In this study, we have investigated the role of the mammalian homologue of aspergillus nidulans NudE, Ndel1, emergently viewed as an integrator of the cytoskeleton, in axon regeneration. Ndel1 was synthesized de novo and upregulated in crushed and transected sciatic nerve axons, and, upon injury, was strongly associated with neuronal form of the intermediate filament (IF) Vimentin while dissociating from the mature neuronal IF (Neurofilament) light chain NF-L. Consistent with a role for Ndel1 in the conditioning lesion-induced neurite outgrowth of Dorsal Root Ganglion (DRG) neurons, the long lasting in vivo formation of the neuronal Ndel1/Vimentin complex was associated with robust axon regeneration. Furthermore, local silencing of Ndel1 in transected axons by siRNA severely reduced the extent of regeneration in vivo. Thus, Ndel1 promotes axonal regeneration; activating this endogenous repair mechanism may enhance neuroregeneration during acute and chronic axonal degeneration.
Vertebrate Lrig3-ErbB Interactions Occur In Vitro but Are Unlikely to Play a Role in Lrig3-Dependent Inner Ear MorphogenesisAbraira, Victoria E.; Satoh, Takunori; Fekete, Donna M.; Goodrich, Lisa V.; Mei, Lin; Department of Neurology; College of Graduate Studies (2010-02-1)Background: The Lrig genes encode a family of transmembrane proteins that have been implicated in tumorigenesis, psoriasis, neural crest development, and complex tissue morphogenesis. Whether these diverse phenotypes reflect a single underlying cellular mechanism is not known. However, Lrig proteins contain evolutionarily conserved ectodomains harboring both leucine-rich repeats and immunoglobulin domains, suggesting an ability to bind to common partners. Previous studies revealed that Lrig1 binds to and inhibits members of the ErbB family of receptor tyrosine kinases by inducing receptor internalization and degradation. In addition, other receptor tyrosine kinase binding partners have been identified for both Lrig1 and Lrig3, leaving open the question of whether defective ErbB signaling is responsible for the observed mouse phenotypes.
Linear Approaches to Intramolecular Forster Resonance Energy Transfer Probe Measurements for Quantitative ModelingBirtwistle, Marc R.; von Kriegsheim, Alexander; Kida, Katarzyna; Schwarz, Juliane P.; Anderson, Kurt I.; Kolch, Walter; GHSU Cancer Center (2011-11-16)Numerous unimolecular, genetically-encoded Forster Resonance Energy Transfer (FRET) probes for monitoring biochemical activities in live cells have been developed over the past decade. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells and tissues, they are an attractive technology for obtaining data to develop quantitative, mathematical models of spatiotemporal signaling dynamics. However, to be useful for such purposes the observed FRET from such probes should be related to a biological quantity of interest through a defined mathematical relationship, which is straightforward when this relationship is linear, and can be difficult otherwise. First, we show that only in rare circumstances is the observed FRET linearly proportional to a biochemical activity. Therefore in most cases FRET measurements should only be compared either to explicitly modeled probes or to concentrations of products of the biochemical activity, but not to activities themselves. Importantly, we find that FRET measured by standard intensity-based, ratiometric methods is inherently non-linear with respect to the fraction of probes undergoing FRET. Alternatively, we find that quantifying FRET either via (1) fluorescence lifetime imaging (FLIM) or (2) ratiometric methods where the donor emission intensity is divided by the directly-excited acceptor emission intensity (denoted Ralt) is linear with respect to the fraction of probes undergoing FRET. This linearity property allows one to calculate the fraction of active probes based on the FRET measurement. Thus, our results suggest that either FLIM or ratiometric methods based on Ralt are the preferred techniques for obtaining quantitative data from FRET probe experiments for mathematical modeling purposes.