Droplet and fibril formation of the functional amyloid Orb2

The functional amyloid Orb2 belongs to the cytoplasmic polyadenylation element binding (CPEB) protein family and plays an important role in long-term memory formation in Drosophila. The Orb2 domain structure combines RNA recognition motifs with low-complexity sequences similar to many RNA-binding proteins shown to form protein droplets via liquid–liquid phase separation (LLPS) in vivo and in vitro. This similarity suggests that Orb2 might also undergo LLPS. However, cellular Orb2 puncta have very little internal protein mobility, and Orb2 forms fibrils in Drosophila brains that are functionally active indicating that LLPS might not play a role for Orb2. In the present work, we reconcile these two views on Orb2 droplet formation. Using fluorescence microscopy, we show that soluble Orb2 can indeed phase separate into protein droplets. However, fluorescence recovery after photobleaching (FRAP) data shows that these droplets have either no or only an extremely short-lived liquid phase and appear maturated right after formation. Orb2 fragments that lack the C-terminal RNA-binding domain (RBD) form fibrils out of these droplets. Solid-state NMR shows that these fibrils have well-ordered static domains in addition to the Gln/His-rich fibril core. Further, we find that full-length Orb2B, which is by far the major component of Orb2 fibrils in vivo, does not transition into fibrils but remains in the droplet phase. Together, our data suggest that phase separation might play a role in initiating the formation of functional Orb2 fibrils.