Droplet and fibril formation of the functional amyloid Orb2

Abstract

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. 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. Orb2 is a cytoplasmic polyadenylation element binding (CPEB) protein that can form functional cross-β (amyloid) fibrils with a regulatory role for long-term memory (LTM) formation in Drosophila (1Si K. Kandel E.R. The role of functional prion-like proteins in the persistence of memory.Cold Spring Harb. Perspect. Biol. 2016; 8a021774Crossref PubMed Scopus (69) Google Scholar). In its monomeric form, it promotes the deadenylation of target messenger mRNA. When aggregating into cross-β fibrils, it becomes an activator of the polyadenylation and thereby the translation of mRNAs resulting in a stabilization of memories past 48 h (2Keleman K. Krüttner S. Alenius M. Dickson B.J. Function of the Drosophila CPEB protein Orb2 in long-term courtship memory.Nat. Neurosci. 2007; 10: 1587-1593Crossref PubMed Scopus (179) Google Scholar, 3Krüttner S. Stepien B. Noordermeer J.N. Mommaas M.A. Mechtler K. Dickson B.J. Keleman K. Drosophila CPEB Orb2A mediates memory independent of its RNA-binding domain.Neuron. 2012; 76: 383-395Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 4Majumdar A. Cesario W.C. White-Grindley E. Jiang H. Ren F. Khan M.R. Li L. Choi E.M.-L. Kannan K. Guo F. Unruh J. Slaughter B. Si K. Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory.Cell. 2012; 148: 515-529Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 5Hervás R. Li L. Majumdar A. Fernández-Ramírez Mdel C. Unruh J.R. Slaughter B.D. Galera-Prat A. Santana E. Suzuki M. Nagai Y. Bruix M. Casas-Tintó S. Menéndez M. Laurents D.V. Si K. et al.Molecular basis of Orb2 amyloidogenesis and blockade of memory consolidation.PLoS Biol. 2016; 14: e1002361Crossref PubMed Scopus (43) Google Scholar). Orb2 has two isoforms, Orb2A and Orb2B, which both share two C-terminal RNA recognition motifs (RRMs), a C-terminal zinc finger, a central Gly-rich region, and a Gln/His-rich domain that forms the core of Orb2 fibrils (see Fig. 1) (2Keleman K. Krüttner S. Alenius M. Dickson B.J. Function of the Drosophila CPEB protein Orb2 in long-term courtship memory.Nat. Neurosci. 2007; 10: 1587-1593Crossref PubMed Scopus (179) Google Scholar, 6Hervás R. Murzin A.G. Si K. Implications of the Orb2 amyloid structure in Huntington’s disease.Int. J. Mol. Sci. 2020; 21: 6910Crossref Scopus (1) Google Scholar). In vivo, Orb2A is of relatively low abundance and only increases in concentration upon synaptic stimulation (7White-Grindley E. Li L. Mohammad Khan R. Ren F. Saraf A. Florens L. Si K. Contribution of Orb2A stability in regulated amyloid-like oligomerization of Drosophila Orb2.PLoS Biol. 2014; 12e1001786Crossref PubMed Scopus (37) Google Scholar). However, its presence is essential for the aggregation of the common isoform Orb2B (3Krüttner S. Stepien B. Noordermeer J.N. Mommaas M.A. Mechtler K. Dickson B.J. Keleman K. Drosophila CPEB Orb2A mediates memory independent of its RNA-binding domain.Neuron. 2012; 76: 383-395Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 4Majumdar A. Cesario W.C. White-Grindley E. Jiang H. Ren F. Khan M.R. Li L. Choi E.M.-L. Kannan K. Guo F. Unruh J. Slaughter B. Si K. Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory.Cell. 2012; 148: 515-529Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Orb2A has nine unique N-terminal residues that can form cross-β fibrils on their own (8Cervantes S.A. Bajakian T.H. Soria M.A. Falk A.S. Service R.J. Langen R. Siemer A.B. Identification and structural characterization of the N-terminal amyloid core of Orb2 isoform A.Sci. Rep. 2016; 6: 38265Crossref PubMed Scopus (20) Google Scholar) and whose deletion or mutation prevents Orb2 aggregation and LTM formation (4Majumdar A. Cesario W.C. White-Grindley E. Jiang H. Ren F. Khan M.R. Li L. Choi E.M.-L. Kannan K. Guo F. Unruh J. Slaughter B. Si K. Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory.Cell. 2012; 148: 515-529Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). The deletion of Orb2A’s C-terminus has no phenotype, whereas its deletion in Orb2B was lethal (3Krüttner S. Stepien B. Noordermeer J.N. Mommaas M.A. Mechtler K. Dickson B.J. Keleman K. Drosophila CPEB Orb2A mediates memory independent of its RNA-binding domain.Neuron. 2012; 76: 383-395Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 9Li L. Sanchez C.P. Slaughter B.D. Zhao Y. Khan M.R. Unruh J.R. Rubinstein B. Si K. A putative biochemical engram of long-term memory.Curr. Biol. 2016; 26: 3143-3156Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The N-terminus of Orb2B is Ser/Gly-rich and of unknown function. Hervás and coworkers recently determined the structure of the Orb2 fibril core, which is located in the Gln/His-domain, using cryo-EM (6Hervás R. Murzin A.G. Si K. Implications of the Orb2 amyloid structure in Huntington’s disease.Int. J. Mol. Sci. 2020; 21: 6910Crossref Scopus (1) Google Scholar). The domain structure of Orb2 that combines low-complexity sequences with RNA-binding domains (RBDs) is reminiscent of a whole class of RNA-binding proteins, such as fused in sarcoma (FUS), that are able to undergo liquid–liquid phase separation (LLPS) (10King O.D. Gitler A.D. Shorter J. The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease.Brain Res. 2012; 1462: 61-80Crossref PubMed Scopus (410) Google Scholar, 11Kato M. Han T.W. Xie S. Shi K. Du X. Wu L.C. Mirzaei H. Goldsmith E.J. Longgood J. Pei J. Grishin N.V. Frantz D.E. Schneider J.W. Chen S. Li L. et al.Cell-free formation of RNA granules: Low complexity sequence domains form dynamic fibers within hydrogels.Cell. 2012; 149: 753-767Abstract Full Text Full Text PDF PubMed Scopus (1113) Google Scholar, 12Li J. McQuade T. Siemer A.B. Napetschnig J. Moriwaki K. Hsiao Y.-S. Damko E. Moquin D. Walz T. McDermott A. Chan F.K.-M. Wu H. The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis.Cell. 2012; 150: 339-350Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar). LLPS is often of functional relevance e.g., for RNA processing in the case of (stress) granules (13Lin Y. Protter D.S.W. Rosen M.K. Parker R. Formation and maturation of phase-separated liquid droplets by RNA-binding Full Text Full Text PDF PubMed Scopus Google Scholar) or for and in X. M. phase separation in and synaptic 2020; Full Text Full Text PDF PubMed Scopus Google Scholar, X. Wu X. Wu H. M. separation the Neurosci. 2020; PubMed Scopus Google Scholar). proteins such as or have to undergo LLPS F. J. R. A liquid to phase transition Full Text Full Text PDF PubMed Scopus Google Scholar, H. S. C.P. A.S. RNA protein phase Full Text Full Text PDF PubMed Scopus Google Scholar). When via LLPS as droplet or puncta formation. relatively can which in the to with droplets and low fluorescence recovery after photobleaching (FRAP) R. A of low complexity Biol. 2016; PubMed Scopus Google Scholar, Rosen M.K. of cellular Mol. Biol. PubMed Scopus Google Scholar). maturation is by protein in the droplet which becomes a protein droplets can in such as or the of cross-β fibril formation F. J. R. A liquid to phase transition Full Text Full Text PDF PubMed Scopus Google Scholar, A. L. S. M. S. J. S. A. M. et phase transition of the protein by Full Text Full Text PDF PubMed Scopus Google Scholar). these suggest that Orb2 also undergo phase which have a role for its or its aggregation into cross-β fibrils, or In the we the of or not Orb2 can undergo phase separation by that it indeed Further, we droplet maturation and fibril formation of Orb2 fragments to the Orb2 droplet and fibril formation. Orb2 can phase separate in we full-length Orb2B and Orb2A and Orb2B fragments the C-terminal which we to as not full-length Orb2A it does not its for in vivo (3Krüttner S. Stepien B. Noordermeer J.N. Mommaas M.A. Mechtler K. Dickson B.J. Keleman K. Drosophila CPEB Orb2A mediates memory independent of its RNA-binding domain.Neuron. 2012; 76: 383-395Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and we not able to it in using the M. A. M. M. of the droplet of proteins in the Sci. S. A. 2020; PubMed Google Scholar) a for phase separation for the of Orb2A and Orb2B the of the the N-terminal residues unique to Orb2A a the residues in This does not to by separation as the in Orb2 can undergo phase we in that a monomeric to as When in Orb2 not phase separate or form droplet we Orb2 into and upon the that Orb2B, and droplet we and fluorescence of protein protein droplets for after these droplets to in and not of the in to many protein droplets. The of and Orb2B droplets and (see Fig. Y. M. of by Sci. 2016; Scopus Google Scholar) that the droplets a of of low we that both and a able to Orb2 droplet formation (see Fig. to droplets are these protein droplets a transition a in which proteins the droplet to a with little to no protein phase separation and its role in protein Mol. Biol. 2020; PubMed Scopus Google Scholar). protein we the of Orb2 droplets using an and the recovery by for our of the Orb2 of within of droplet formation. that Orb2 is static within droplets after phase separation these we on the of Orb2B droplets. The not after the In we of Orb2B droplets with droplet and many proteins, the of a droplet of the droplets with the that Orb2B becomes static within droplets relatively after phase a we a and for which we recovery in fluorescence after similar to on droplet A. L. S. M. S. J. S. A. M. et phase transition of the protein by Full Text Full Text PDF PubMed Scopus Google Scholar). proteins, droplet maturation is by cross-β fibril formation R. A of low complexity Biol. 2016; PubMed Scopus Google Scholar). Orb2 droplets and in cross-β fibrils, we Orb2 droplets using fluorescence microscopy, and and of and Orb2B in are shown in In the case of the droplets h in our fluorescence which with fibrils out a as by droplets into fibrils and droplets 48 we in fluorescence microscopy, which with fibrils in our In Orb2B droplets to form fibrils, but that of droplets similar to shown in the of and droplets to the maturation of these droplets into cross-β fibrils When using fibrils out a common a In fibrils can both fibril not show structure or and an of an of droplet to fibril we fluorescence and for Orb2 in both and not in fluorescence in for of the In and an in fluorescence in a for cross-β fibril formation. In Orb2B only a in in with to droplets. The of and in the h and for after a for was after This is with the of and droplets. The in for with the of fibrils as in our that the fibrils by The of Orb2B our with the formation of relatively droplets. separation of and to the formation of cross-β fibrils. we does the structure of these fibrils with the cryo-EM structure of Orb2 fibrils Drosophila (6Hervás R. Murzin A.G. Si K. Implications of the Orb2 amyloid structure in Huntington’s disease.Int. J. Mol. Sci. 2020; 21: 6910Crossref Scopus (1) Google Scholar) and the structure of fibrils we (8Cervantes S.A. Bajakian T.H. Soria M.A. Falk A.S. Service R.J. Langen R. Siemer A.B. Identification and structural characterization of the N-terminal amyloid core of Orb2 isoform A.Sci. Rep. 2016; 6: 38265Crossref PubMed Scopus (20) Google we and and fibrils via phase separation for NMR of these fibrils are shown in static protein domains that in the case of cross-β fibrils often with the fibril core. in are to dynamic protein that are often the static fibril core. the both and fibrils have static and dynamic The of the of these two fibril relatively indicating that both fibrils have similar also show with a fibril core. However, the the that their fibril core are not the The of that these fibrils have dynamic the residues in the fibril core, we that the residues in the fibrils, which often with its core (8Cervantes S.A. Bajakian T.H. Soria M.A. Falk A.S. Service R.J. Langen R. Siemer A.B. Identification and structural characterization of the N-terminal amyloid core of Orb2 isoform A.Sci. Rep. 2016; 6: 38265Crossref PubMed Scopus (20) Google Scholar, A.B. C. T. M. 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In we many residues that are not of the Gln/His-rich core, and The of these is with an The that the two fibrils that are very indicating a similar core we able to in the of that not in are with in and and these not to an fibril core located the N-terminal unique to as is not in are on the of these to the and structure of the static domains and their the in our Orb2 fibrils with in of and two proteins that form fibrils with a shows the of of and fibrils, both of which phase This shows that the in not to of the two major A and in the fibril Langen R. Siemer A.B. Solid-state on the static and dynamic domains of PubMed Scopus Google Scholar, Langen A. Siemer A.B. formation and structure of PubMed Scopus Google Scholar, R. H. D. H. D. E. S. R. M. M. characterization of fibrils by NMR Mol. Biol. PubMed Scopus Google Scholar, M. R. R. The N-terminus is in amyloid PubMed Scopus Google Scholar, C. K. M. R. amyloid core and domain in fibrils determined by 2014; PubMed Scopus Google Scholar, K. 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Khan M.R. Li L. Choi E.M.-L. Kannan K. Guo F. Unruh J. Slaughter B. Si K. Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory.Cell. 2012; 148: 515-529Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). reconcile these two views by that Orb2 phase separation does In to proteins that undergo maturation liquid to droplets with little to no protein mobility, the Orb2 droplets we either a liquid phase or is short-lived that we not able to In Orb2 droplets are similar to by the complex protein M. A. K. C. M. structure in an fibril with Sci. S. A. PubMed Scopus Google the protein in E. M. M. M. R. S. D. of a cellular 2016; Full Text Full Text PDF PubMed Scopus Google Scholar) or D.E. M. M. C. T. M. S. A. L. et to by protein 2016; Full Text Full Text PDF PubMed Scopus Google Scholar, E. and of Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). In which can form protein droplets that into fibrils with core similar to forms F. J. R. A liquid to phase transition Full Text Full Text PDF PubMed Scopus Google Scholar). In our phase separation is a to soluble into amyloid fibrils. that a of Orb2A forms cross-β fibrils in (8Cervantes S.A. Bajakian T.H. Soria M.A. Falk A.S. Service R.J. Langen R. Siemer A.B. Identification and structural characterization of the N-terminal amyloid core of Orb2 isoform A.Sci. Rep. 2016; 6: 38265Crossref PubMed Scopus (20) Google Scholar). However, the fibril core of was not located in the domain but the very N-terminus unique to In fragments not in and required phase separation for fibril formation. Further, NMR of fibrils are with the presence of the fibril core in the cryo-EM structure of Orb2 fibrils (6Hervás R. Murzin A.G. Si K. Implications of the Orb2 amyloid structure in Huntington’s disease.Int. J. Mol. Sci. 2020; 21: 6910Crossref Scopus (1) Google Scholar). NMR also show that are static domains in and fibrils not by the fibril core. domains and and This does not to these domains within the is little that are located in the residues of Orb2A the that is for fibrils by these residues (8Cervantes S.A. Bajakian T.H. Soria M.A. Falk A.S. Service R.J. Langen R. Siemer A.B. Identification and structural characterization of the N-terminal amyloid core of Orb2 isoform A.Sci. Rep. 2016; 6: 38265Crossref PubMed Scopus (20) Google Scholar) was our are on the of these In addition to fibril and also form droplets that in independent of fibrils are whereas fibrils show little to the Orb2 play a role in droplet and fibril the NMR of fibrils with the NMR of and This is of the fibril core of Orb2 is reminiscent of the fibril core of and the core of of the of and fibril that the of the residues in both fibril is which in with the that the Orb2 fibril core is an (6Hervás R. Murzin A.G. Si K. Implications of the Orb2 amyloid structure in Huntington’s disease.Int. J. Mol. Sci. 2020; 21: 6910Crossref Scopus (1) Google whereas is not T. Langen R. and domain of Biol. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar). This might also the of of fibrils B. L. Y. A.B. R. A. Solid-state NMR of the amyloid core of full-length NMR 2014; PubMed Scopus Google Scholar) our fibrils shown to form F. D. Chen B. R. of have independent of PubMed Scopus Google Scholar). the to the in our was by the cryo-EM structure of the Orb2 fibril core, that our fibrils are similar in structure to in Orb2 fragments the C-terminal form cross-β fibrils after droplet full-length Orb2B remains in a of which but transition into cross-β fibrils. This is with in vivo that Orb2B form functional that the translation of target (4Majumdar A. Cesario W.C. White-Grindley E. Jiang H. Ren F. Khan M.R. Li L. Choi E.M.-L. Kannan K. Guo F. Unruh J. Slaughter B. Si K. Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory.Cell. 2012; 148: 515-529Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). forms fibrils via droplet the C-terminal plays an important role in fibril formation of has for the RNA-binding protein which can phase separate in the presence of RNA but forms its is H. S. C.P. A.S. RNA protein phase Full Text Full Text PDF PubMed Scopus Google Scholar). The cross-β fibril of Orb2 has shown to a functional important in LTM (6Hervás R. Murzin A.G. Si K. Implications of the Orb2 amyloid structure in Huntington’s disease.Int. J. Mol. Sci. 2020; 21: 6910Crossref Scopus (1) Google the role of phase separation in LTM is that phase separation might the that fibril formation of Orb2 in the Orb2 aggregation and LTM in Drosophila on the and thereby concentration of cellular Orb2A via (7White-Grindley E. Li L. Mohammad Khan R. Ren F. Saraf A. Florens L. Si K. Contribution of Orb2A stability in regulated amyloid-like oligomerization of Drosophila Orb2.PLoS Biol. 2014; 12e1001786Crossref PubMed Scopus (37) Google Scholar). This in concentration phase However, many that Orb2 phase separation in the formation via LLPS has for proteins in neurodegenerative F. J. R. A liquid to phase transition Full Text Full Text PDF PubMed Scopus Google Scholar, A. L. S. M. S. J. S. A. M. et phase transition of the protein by Full Text Full Text PDF PubMed Scopus Google Scholar, Shorter J. phase in neurodegenerative Mol. Res. PubMed Scopus Google Scholar). LLPS might important and a for fibril formation of functional In our data droplet formation as a for Orb2 fibril formation in This to in on our to monomeric Orb2 and its phase our a functional Orb2 protein complex in and the structural of its The sequences for and for in E. and into with a C-terminal by Orb2B was a by the of into a The Orb2B was in E. whereas and in or A was to with the and and The of was into with the and to an of was with the addition of and for an h via in a for and or for to in and and of using a and the was for using a The was and a The protein The was with and was The protein was with and in was using the protein of and a The protein was using and the protein or in liquid and was and as Langen A. Siemer A.B. formation and structure of PubMed Scopus Google Scholar, A. Langen J. Langen R. The in aggregation in and on via Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). and was in in and in a using The protein was in and such that the protein concentration was The was for these forms fibrils via phase fibrils by for in a The fibrils with into a NMR that for either in a that or into a with using a The protein with either or with Orb2B and with to the lack of residues in the protein it was with The and of these are and droplet protein was either into and or into by the addition of or by and using a with also on after the fluorescence by a for to a of protein droplets and 48 h into the using J. E. M. T. S. C. S. B. K. et for 2012; PubMed Scopus Google Scholar, J. for the of PubMed Scopus Google Scholar) the was on protein to into with using a and using with a with an of to in droplets of C.P. in phase-separated using fluorescence recovery after J. 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