Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling

Abstract

Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin's high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method's sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism. Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin's high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method's sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism. Characterizing the interactomes of specific proteins, and the proteome profiles of subcellular structures, cells, and tissues, provides a powerful entry point to probe molecular function. Several methods designed to highlight protein–protein interactions (PPIs) have proven useful, including yeast-two-hybrid (Y2H), affinity purification, and phage display (1Qin W. Cho K.F. Cavanagh P.E. Ting A.Y. Deciphering molecular interactions by proximity labeling.Nat. Methods. 2021; 18: 133-143Crossref PubMed Scopus (28) Google Scholar, 2Kim D.I. Roux K.J. Filling the void: Proximity-based labeling of proteins in living cells.Trends Cell Biol. 2016; 26: 804-817Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 3Varnaitė R. MacNeill S.A. Meet the neighbors: Mapping local protein interactomes by proximity-dependent labeling with BioID.Proteomics. 2016; 16: 2503-2518Crossref PubMed Scopus (82) Google Scholar, 4Trinkle-Mulcahy L. Recent advances in proximity-based labeling methods for interactome mapping.F1000Research. 2019; 8Crossref PubMed Scopus (55) Google Scholar, 5Rees J.S. Li X.W. Perrett S. Lilley K.S. Jackson A.P. Protein neighbors and proximity proteomics.Mol. Cell. Proteomics. 2015; 14: 2848-2856Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). However, each method has limitations that can include high false-positive rates, poor detection of transient or weak interactors, a low signal-to-noise ratio when detecting PPIs in specific cell types or subcellular compartments, artifacts created during tissue homogenization, and competing requirements for solubilizing proteins while keeping complexes intact (1Qin W. Cho K.F. Cavanagh P.E. Ting A.Y. Deciphering molecular interactions by proximity labeling.Nat. Methods. 2021; 18: 133-143Crossref PubMed Scopus (28) Google Scholar, 2Kim D.I. Roux K.J. Filling the void: Proximity-based labeling of proteins in living cells.Trends Cell Biol. 2016; 26: 804-817Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Proximity-labeling methods overcome many of these limitations (1Qin W. Cho K.F. Cavanagh P.E. Ting A.Y. Deciphering molecular interactions by proximity labeling.Nat. Methods. 2021; 18: 133-143Crossref PubMed Scopus (28) Google Scholar, 6Xu Y. Fan X. Hu Y. In vivo interactome profiling by enzyme-catalyzed proximity labeling.Cell Biosci. 2021; 11: 1-9Crossref PubMed Scopus (5) Google Scholar, 7Samavarchi-Tehrani P. Samson R. Gingras A.C. Proximity dependent biotinylation: Key enzymes and adaptation to proteomics approaches.Mol. Cell. Proteomics. 2020; 19: 757-773Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 8Gingras A.C. Abe K.T. Raught B. Getting to know the neighborhood: Using proximity-dependent biotinylation to characterize protein complexes and map organelles.Curr. Opin. Chem. Biol. 2019; 48: 44-54Crossref PubMed Scopus (95) Google Scholar) and have allowed the proteomes of subcellular compartments (1Qin W. Cho K.F. Cavanagh P.E. Ting A.Y. Deciphering molecular interactions by proximity labeling.Nat. Methods. 2021; 18: 133-143Crossref PubMed Scopus (28) Google Scholar) and weak or transient PPIs to be characterized in vivo (1Qin W. Cho K.F. Cavanagh P.E. Ting A.Y. Deciphering molecular interactions by proximity labeling.Nat. Methods. 2021; 18: 133-143Crossref PubMed Scopus (28) Google Scholar). Proximity labeling fuses a protein of interest to an enzyme domain that promiscuously tags proteins in its vicinity with a biochemical handle. This handle allows selective recovery of tagged proteins, which can then be identified by mass spectrometry (MS) (1Qin W. Cho K.F. Cavanagh P.E. Ting A.Y. Deciphering molecular interactions by proximity labeling.Nat. Methods. 2021; 18: 133-143Crossref PubMed Scopus (28) Google Scholar, 2Kim D.I. Roux K.J. Filling the void: Proximity-based labeling of proteins in living cells.Trends Cell Biol. 2016; 26: 804-817Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 7Samavarchi-Tehrani P. Samson R. Gingras A.C. Proximity dependent biotinylation: Key enzymes and adaptation to proteomics approaches.Mol. Cell. Proteomics. 2020; 19: 757-773Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The enzyme domains most widely used for proximity labeling in living cells and organisms are engineered variants of the E. coli biotin ligase BirA, e.g., BioID and TurboID, or of ascorbate peroxidase, e.g., APEX. In vivo applications in animals have, however, been relatively limited (9Uezu A. Kanak D.J. Bradshaw T.W.A. Soderblom E.J. Catavero C.M. Burette A.C. Weinberg R.J. Soderling S.H. Identification of an elaborate complex mediating postsynaptic inhibition.Science. 2016; 353: 1123-1129Crossref PubMed Scopus (132) Google Scholar, 10Dingar D. Kalkat M. Chan P.K. Srikumar T. Bailey S.D. Tu W.B. Coyaud E. Ponzielli R. Kolyar M. Jurisica I. Huang A. Lupien M. Penn L.Z. Raught B. BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors.J. Proteomics. 2015; 118: 95-111Crossref PubMed Scopus (76) Google Scholar) and required genetic modifications to alter cuticle permeability in the nematode (11Reinke A.W. Balla K.M. Bennett E.J. Troemel E.R. Identification of microsporidia host-exposed proteins reveals a repertoire of rapidly evolving proteins.Nat. Commun. 2017; 8: 1-11Crossref PubMed Scopus (39) Google Scholar, 12Reinke A.W. Mak R. Troemel E.R. Ben E.J. In vivo mapping of tissue- and subcellular-specific proteomes in Caenorhabditis elegans.Sci. Adv. 2017; 3: 1-12Crossref Scopus (30) Google Scholar) and tissue dissection to increase H2O2 delivery to tissues (13Chen C.L. Hu Y. Udeshi N.D. Lau T.Y. Wirtz-Peitz F. He L. Ting A.Y. Carr S.A. Perrimon N. Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 12093-12098Crossref PubMed Scopus (85) Google Scholar) or pretreatment of live samples with detergent to increase biotin-phenol permeability in the fly (14Mannix K.M. Starble R.M. Kaufman R.S. Cooley L. Proximity labeling reveals novel interactomes in live Drosophila tissue.Development. 2019; 146dev176644Crossref PubMed Google Scholar). Caenorhabditis elegans has proven a useful workhorse to investigate metazoan biology, offering powerful genetics, in vivo cell biology, an anatomy described at electron micrograph resolution, and a defined number of cells whose gene expression can be profiled at single cell resolution (15Packer J.S. Zhu Q. Huynh C. Sivaramakrishnan P. Preston E. Dueck H. Stefanik D. Tan K. Trapnell C. Kim J. Waterston R.H. Murray J.I. A lineage-resolved molecular atlas of C. elegans embryogenesis at single-cell resolution.Science. 2019; 365eaax1971Crossref PubMed Scopus (103) Google Scholar, 16White J.G. Southgate E. Thomson J.N. Brenner S. The structure of the nervous system of the nematode Caenorhabditis elegans.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1986; 314: 1-340Crossref PubMed Google Scholar, 17Taylor S.R. Santpere G. Reilly M. Glenwinkel L. Poff A. McWhirter R. Xu C. Weinreb A. Basavaraju M. Cook S.J. Barrett A. Abrams A. Vidal B. Cros C. Rafi I. et al.Expression profiling of the mature C. elegans nervous system by single-cell RNA-sequencing.bioRxiv. 2019; https://doi.org/10.1101/737577Crossref Scopus (0) Google Scholar, 18Lorenzo R. Onizuka M. M. P. single-cell with a molecular atlas for Caenorhabditis elegans 2020; 48: Google Scholar). The ability to probe protein complexes in vivo using proximity labeling a to in this C. elegans advantages for proximity protein can be and functionally of can be and proximity labeling can be to specific cell using R. Onizuka M. M. P. single-cell with a molecular atlas for Caenorhabditis elegans 2020; 48: Google Scholar, W. X. U. Huang He H. Li R. system for gene expression in C. 2020; Full Text Full Text PDF PubMed Scopus Google Scholar, T. S. of expression in Caenorhabditis PubMed Scopus Google Scholar). proteins can be rapidly using gene or e.g., using J. C. The elegans 2019; PubMed Scopus Google Scholar). C. elegans at BioID at Roux K.J. of BioID and TurboID for 2020; Scopus Google for in the To is BioID in C. elegans. However, expressed in specific C. elegans tissues can proteins to an a of U. A. K.M. A. N. R. E. a for PubMed Scopus Google Scholar). The protein can then be affinity and proteins identified by S. J. C. T.Y. M. T. M. S. J. et protein in Caenorhabditis elegans identifies novel partners of Biol. 2016; 14: PubMed Scopus Google Scholar). et (11Reinke A.W. Balla K.M. Bennett E.J. Troemel E.R. Identification of microsporidia host-exposed proteins reveals a repertoire of rapidly evolving proteins.Nat. Commun. 2017; 8: 1-11Crossref PubMed Scopus (39) Google Scholar, 12Reinke A.W. Mak R. Troemel E.R. Ben E.J. In vivo mapping of tissue- and subcellular-specific proteomes in Caenorhabditis elegans.Sci. Adv. 2017; 3: 1-12Crossref Scopus (30) Google Scholar) expressed or in C. elegans tissues and characterized the has however, been used to PPIs in The is labeling proteins the protein and to Hu R. K. Ting A.Y. M. Hu R. K. Ting A.Y. M. An to protein in living 2017; Full Text Full Text PDF PubMed Scopus Google Scholar). characterized tissue proteomes in C. elegans by A. S. in Caenorhabditis Natl. Acad. Sci. U. S. A. 2015; 112: PubMed Scopus Google Scholar, S. Y. T. H. W. M. in Caenorhabditis elegans.Sci. 2020; 1-9Crossref PubMed Scopus Google Scholar). can with that this can be into proteins during the to be and proteins affinity for mass the proteome of tissues, including however, be used to of BioID that are TurboID and have been using Udeshi N.D. T. Carr S.A. Perrimon N. Ting A.Y. proximity labeling in living cells and organisms with PubMed Scopus Google Scholar). in C. elegans in biotinylation in the with the by TurboID Udeshi N.D. T. Carr S.A. Perrimon N. Ting A.Y. proximity labeling in living cells and organisms with PubMed Scopus Google Scholar). However, the interactome or for C. elegans. A used TurboID to characterize the interactome of the protein this protein in the tissue of the the gut, and to optimize the protocol A. X. K. C. Ting A.Y. Proximity labeling at reveals and as 2020; Scopus (0) Google Scholar). we optimize a protocol for in C. elegans. We TurboID in C. elegans tissues and in the of AFD We TurboID into which encodes a presynaptic active zone protein. We characterize tissue proteomes and highlight proteins. TurboID to AFD neurons the proteins and and previously uncharacterized proteins that we are selectively expressed in AFD. The samples are in the synaptic proteins protein and previously uncharacterized proteins, which we at that proximity labeling can identify the proteome of a of neurons and of a synaptic protein in C. elegans in To the to which TurboID proteins in C. elegans we animals a protein in tissues We protein biotinylation in the with In the of animals showed a increase in proteins with biotin protein biotinylation in animals the To optimize biotin to worm tissues, we animals with biotin for using biotinylation in neurons as a A to protein labeling To identify an biotin for protein we animals with of biotin for We a increase in biotinylation in with biotin biotin to increase biotinylation We used the E. coli biotin as a for of D. A. E. K. R. A. with in gene expression in nematode and 3: PubMed Scopus Google to the biotin to addition of for of the during which biotinylation and expression of biotinylation that TurboID is in C. elegans We a protocol to and affinity proteins C. elegans A advantage of with is that can be under denaturing the biotin is This allowed to solubilization of proteins We achieved protein using a and to of Using of to for with of biotin for at and with protein worm samples and to biotin for affinity The with streptavidin to affinity proteins showed of the of proteins the using our affinity protocol extensive and we the affinity-purified proteins by the proteins by and by Using a of at two we identified proteins expressed in one or tissues B and Using tissue D. Chan J. for C. elegans 2016; PubMed Scopus Google we for in the of proteins we identified as to each with the tissue in each the of the method for tissues In we a the two at the of with we identified most proteins in the by the and cells A.W. Mak R. Troemel E.R. Ben E.J. In vivo mapping of tissue- and subcellular-specific proteomes in Caenorhabditis elegans.Sci. Adv. 2017; 3: 1-12Crossref Scopus (30) Google Scholar). proteins at in one tissue tissues and proteins in one tissue identified by when TurboID to neurons expressed proteins, as and proteins expressed in of as the which is known to be expressed in and which is expressed in of neurons samples in and samples in and and samples and In we identified for many proteins whose expression previously uncharacterized of these proteins we the expression by our by The the expression by the method's The C. elegans nervous system of with most of a single of neurons that J.G. Southgate E. Thomson J.N. Brenner S. The structure of the nervous system of the nematode Caenorhabditis elegans.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1986; 314: 1-340Crossref PubMed Google Scholar). We our protocol to characterize proteins expressed in a single of To test we expressed in the AFD of using the of these animals a biotinylation that in of mass spectrometry for affinity-purified animals the showed these samples for proteins or selectively expressed in AFD neurons when with or animals a and proteins the and and the and identified proteins in the TurboID samples with the TurboID and To if these proteins selectively expressed in AFD we and expressed in at low expressed in a of neurons that AFD of proteins, identified by mass with gene expression identified by D. R. S. M. The repertoire of Caenorhabditis elegans neurons and its in Biol. Cell. 2016; Scopus (28) Google Scholar). that TurboID is a tool to map the proteome of specific neurons in C. elegans. We if TurboID can highlight the interactome of a specific C. elegans protein expressed at endogenous We the synaptic protein an of is expressed the nervous system and to the presynaptic active in proximity to presynaptic proteins as and The presynaptic active Full Text Full Text PDF PubMed Scopus Google Scholar). To have endogenous of we knocked into the elks-1 using to the and in synapses of animals an biotinylation with However, mass spectrometry of proteins this of known synaptic proteins including and the protein We by mass spectrometry for in and animals the nervous and or nervous system and tissues We identified previously uncharacterized proteins as in samples and and We expressed for of these proteins exclusively in the AFD and showed that with at presynaptic active zones AFD is known to with J.G. Southgate E. Thomson J.N. Brenner S. The structure of the nervous system of the nematode Caenorhabditis elegans.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1986; 314: 1-340Crossref PubMed Google Scholar, A. G. K. synaptic partners cell and synapses in living nervous Full Text Full Text PDF PubMed Scopus Google Scholar) and with our that proximity labeling is an method to protein in C. elegans at endogenous with and We optimize the proximity-dependent protein labeling for C. elegans, and that single proteomics and of the interactome of a synaptic protein expressed at endogenous We that TurboID to be to C. elegans tissues and identify proteins expressed in at one We to identify proteins expressed exclusively in the AFD neurons and previously uncharacterized proteins that are in the vicinity of Proximity labeling methods as TurboID the of the protein. In C. elegans, many that the protein of This can to protein the of identified by proximity proteins at and subcellular of the be to at using identified many proteins to be in the vicinity of include known of in the presynaptic active zone of C. elegans We characterize proteins, and in proteins by and are is to be an protein to and and known as is a for and Cho H. S. A. I. J. E. E. S. et in the gene to a Commun. 2019; PubMed Scopus (5) Google Scholar) and is at postsynaptic and the protein L. and protein in PubMed Scopus Google Scholar). are with this of proteins have of with the presynaptic in neurons Weinberg R.J. of protein in the PubMed Scopus Google and presynaptic proteins with including the Cho H. S. A. I. J. E. E. S. et in the gene to a Commun. 2019; PubMed Scopus (5) Google Scholar). is to a protein that is known this function. is to to and to is known this function. animals an the that is a synaptic protein. be to investigate or has a in synaptic protein that our TurboID is to is the C. elegans of the with the and at presynaptic L. N. and the and of in PubMed Scopus Google Scholar) and optimize of synaptic at the active zone L. N. with a of active zone proteins to optimize the of synaptic in PubMed Scopus (5) Google Scholar). proteins identified in the proximity labeling are We of these proteins be expressed in we of the including the the protein the and T. H. J. Caenorhabditis elegans to and is required for of and in Biol. Cell. 2017; PubMed Google Scholar) in and in that these proteins are expressed in by the to be for using with tagged An for TurboID is the delivery of biotin or biotin In cultured cells TurboID or biotin for as as biotinylation Udeshi N.D. T. Carr S.A. Perrimon N. Ting A.Y. proximity labeling in living cells and organisms with PubMed Scopus Google Scholar). In C. elegans, biotin to with coli or is to TurboID are required to protein biotinylation our We that a with biotin is for TurboID in worm neurons in the C. elegans is E. coli as a E. coli to provides the of biotinylation by be to in a biotin for e.g., or and protein using is required to of the C. elegans proteome however, of the proteins for which we to proteins. protein is a in mass proteins to a of In TurboID is biotinylation be in to the recovery of proteins when TurboID is to of C. elegans cells. the proteome in TurboID as by and A A A the most proteins in our in including and we expressed TurboID in C. elegans tissues, these for of identified in We to tags into each of these and using to by affinity purification, the of proximity when the protein is expressed in a number of cells or at low extensive and of denaturing streptavidin samples the mass identifies many proteins when we our affinity protocol to animals that proteins be expressed at high to to our be endogenous biotinylation of many proteins the highly and A is to a of In our that TurboID well in a of in the TurboID be a and useful tool for the C. elegans to map the proteomes of specific cells and subcellular structures and to characterize the interactomes of proteins of at nematode growth with the biotin E. coli C. elegans S. The of Caenorhabditis PubMed Google Scholar). C. elegans used in this the The is a that tagged with TurboID for C. elegans S. S. S. A. S. H. of protein expression in C. Methods. 8: PubMed Scopus Google Scholar) by and an a and two C. elegans using a which a of and by using that a The into of a The TurboID is at for and C. elegans by and into of a The vectors these each with the a the or and in an The expression vectors into the of at a of for and C. elegans by and into a for a by and into a The vectors with entry and a in an elks-1 a C. elegans and into a with and and a in an expression vectors into the of at a of animals an and to The in a with and to The then to In of the and to a many of the and animals the and for in the to at elks-1 and used of C. elegans proteins using a with or using the The in W. of used to the used in the and with of of C. elegans E. coli at or in at in with and E. coli for the in and the and with The samples then for at for for at at and the to a using for at with and for at with or streptavidin or with then with The or used for this in in in in Cell using the or C. elegans and the to with E. coli to of The animals at or in at in with and E. coli for the in and allowed to the the two of with and to one of The animals allowed to and then to to worm A used to C. elegans to a which then at by the of a and a at the to the at the of the SDS and to the to of and of SDS and The a and at for the samples for with the two sonication used a probe and an of The samples to sonication and to using a urea, The samples then at for at and the the and to a with SDS and urea, with by at for the of then the and by at for the to The using Protein in the samples using protein with streptavidin of C. elegans protein in by in the and using to while we to the of protein to in the mass spectrometry samples with in a and in a at using a to the and a and the to protein with SDS and with to each by biotin to in at for to biotin and the with biotin to a of to for at the and and proteins to a protein of a protein which then with for The into and for mass spectrometry the proteins for mass using the system the protein in a well of a with and with and with proteins with at The in The by using an to a of A the to an using a of to The a with a mass in with an with a of by at at a of using a of for and a to increase of then in S. T. J. The for mass 2016; 11: PubMed Scopus Google Scholar) a the C. elegans proteome and In We used which at the of and to for mass at as modifications as well as two modifications and or protein to for used in the and to then in using the a to to The used to the the a as well as of and at protein to The used the nematode C. elegans, for which is for and an and for The mass spectrometry proteomics have been to the the at with the of C. elegans synapses by proximity We have the at The to the is of the in the of our are in the are and the for each are the This The that have of interest with the of this We for the and of for and for the of mass of worm The biotin E. coli of and to by and and entry by of a of M. S. and M. B. M. A. and S. B. M. A. and S. B. M. A. and M. B. M. S. S. M. F. M. and A. N. M. A. and S. B. M. B. M. B. M. A. and S. B. M. A. M. S. S. M. and M. B. and This by an and a to M. B. and an to M. A.

Topics & Concepts