Dingal, P. C. D. P. | Search bioRxiv

bioRxiv2021

Regulation of Vg1 biogenesis during mesendoderm induction

The TGF-beta signals Vg1 (Dvr1/Gdf3) and Nodal form heterodimers to induce vertebrate mesendoderm. The Vg1 proprotein is a monomer retained in the endoplasmic reticulum (ER) and is processed and secreted upon heterodimerization with Nodal, but the mechanisms underlying Vg1 biogenesis are largely elusive. Here we clarify the mechanisms underlying Vg1 retention, processing, secretion and signaling, and introduce a Synthetic Processing (SynPro) system that enables the programmed cleavage of ER-resident and extracellular proteins. First, we find that Vg1 can be processed by intra- or extracellular proteases. Second, Vg1 can be processed without Nodal but requires Nodal for secretion and signaling. Third, Vg1-Nodal signaling activity requires Vg1 processing, whereas Nodal can remain unprocessed. Fourth, Vg1 employs exposed cysteines, glycosylated asparagines, and BiP chaperone-binding motifs for monomer retention in the ER. These observations suggest two mechanisms for rapid mesendoderm induction: chaperone-binding motifs help store Vg1 as an inactive but ready-to-heterodimerize monomer in the ER, and the flexibility of Vg1 processing location allows efficient generation of active heterodimers both intra- and extracellularly. These results establish SynPro as a new in vivo processing system and define molecular mechanisms and motifs that facilitate the generation of active TGF-beta heterodimers. SignificanceThe TGF-beta family members Nodal and Vg1 are the major inducers of mesendoderm formation during vertebrate embryogenesis. We previously established that the Vg1 proprotein is retained in the endoplasmic reticulum (ER), and that Nodal and Vg1 form heterodimers to pattern the early embryo. However, the mechanisms underlying the retention, processing, secretion, and signaling of Vg1 have been unclear. We found two mechanisms that embryos use to efficiently generate active Nodal-Vg1 heterodimers: (1) Vg1 employs its chaperone-binding motifs to ensure its retention as a ready-to-heterodimerize monomer in the ER; (2) Using a newly devised Synthetic Processing (SynPro) System, we found that Vg1 must be processed for signaling to occur, but its processing location is flexible.

developmental biology↗

bioRxiv2017

Engineering Cell Sensing and Responses Using a GPCR-Coupled CRISPR-Cas System

G-protein coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes, and detects a wide array of physiological cues in the human body. We describe a new molecular device that couples CRISPR-Cas9 programmed genome regulation to natural and synthetic extracellular signals via GPCRs. The design of our synthetic device, named CRISPR ChaCha, displays superior performance over an architecture proposed by the previously reported Tango system. Using a parsimonious mathematical model and gene-reporter assays, we find that CRISPR ChaCha can recruit and activate multiple Cas9 molecules for each GPCR molecule. We also characterize key molecular features that modulate CRISPR ChaCha performance. We adopt the design to diverse GPCRs that sense synthetic and natural ligands including chemokines, mitogens, and fatty acids, and observe efficient conversion of signals to customizable genetic programs in mammalian cells, including regulation of endogenous genes. The new class of CRISPR-coupled GPCRs provides a robust and efficient platform for engineering cells with novel behaviors in response to the diverse GPCR ligand repertoire.

synthetic biology↗