Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells
Macromolecular interactions between and amongst proteins and organelles mediate a considerable amount of biochemical signaling processes . A principal method of testing the physiological significance of such interactions is to drive their association with a user-supplied stimulus such as light or drugs . Typically , two different components , each fused to a specific protein , come together ( ‘heterodimerize’ ) to reconstitute a given protein-protein interaction following addition of a small molecule ( DeRose et al . , 2013; Putyrski and Schultz , 2012; Spencer et al . , 1993 ) or upon light illumination ( Losi et al . , 2018; Rost et al . , 2017 ) . Light offers much greater spatial and temporal resolution than drugs , and as such , optogenetic dimerizers are generally used to probe phenomena at cellular and subcellular scales . At the organism scale , light is much less invasive but suffers from penetration issues . Photodimerizers have been successfully used to manipulate a variety of cellular processes , including signaling networks ( Gasser et al . , 2014; Grusch et al . , 2014; Guglielmi et al . , 2015; Idevall-Hagren et al . , 2012; Toettcher et al . , 2013; Toettcher et al . , 2011; Wu et al . , 2009 ) organelle trafficking ( van Bergeijk et al . , 2015; Duan et al . , 2015 ) , nuclear import/export ( Lerner et al . , 2018; Niopek et al . , 2016; Niopek et al . , 2014 ) , cytoskeletal dynamics ( van Haren et al . , 2018 ) , and phase separation ( Bracha et al . , 2018; Dine et al . , 2018; Shin et al . , 2017 ) , among others . Both natural and synthetic photoswitches ( Guntas et al . , 2015; Losi et al . , 2018; Lungu et al . , 2012; Rost et al . , 2017; Strickland et al . , 2012 ) have been used for these studies , each with its own advantages and drawbacks . Limitations of existing systems include necessity of adding exogenous cofactors ( Levskaya et al . , 2009 ) , large size adversely affecting function of targeted proteins ( Kaberniuk et al . , 2016; Kennedy et al . , 2010; Levskaya et al . , 2009; Yazawa et al . , 2009 ) , non-trivial levels of basal dimerization in the dark ( Guntas et al . , 2015; Hallett et al . , 2016; Nijenhuis et al . , 2020; Zimmerman et al . , 2016 ) , poor light-dependent dimerization efficiency ( Kawano et al . , 2015; Strickland et al . , 2012 ) , and improper homo- , instead of heterodimerization ( Bugaj et al . , 2013; Che et al . , 2015; Duan et al . , 2017; Taslimi et al . , 2016 ) . One popular photodimerizer pair is ‘Magnets’ , engineered from the Neurospora crassa Vivid photoreceptor , which comprises an N-terminal Ncap domain responsible for homodimerization and a C-terminal light-oxygen-voltage-sensing ( LOV ) domain ( Kawano et al . , 2015 ) . Magnets employ the ubiquitous cofactor flavin adenine dinucleotide ( FAD ) as the light-sensing moiety . The Magnets pair was engineered from the Vivid homodimer by introducing complementary charges , giving rise to nMag ( negative Magnet ) and pMag ( positive Magnet ) . The two Magnets components are quite small ( 150 aa ) for photodimerizers , exhibit relatively fast association and dissociation kinetics , and function when fused to a broad range of proteins , including peripheral and intrinsic membrane proteins ( Benedetti et al . , 2018; Kawano et al . , 2016; Kawano et al . , 2015 ) . Furthermore , heterodimerization of Magnets requires light-dependent activation of both components , rather than just one . This property results in low levels of background activity and allows induction of dimer formation with single-wavelength excitation in small cytoplasmic volumes ( Benedetti et al . , 2018 ) . However , the Magnets system has two prominent shortcomings . First , the low thermodynamic stability of the Magnets components precludes their proper expression and folding at 37°C . Thus , they cannot be used in mammals . When used in cultured mammalian cells they require a preincubation at low temperature ( 28°C ) for 12 hr to allow expression and folding . Second , as the Magnets components heterodimerize with low efficiency , robust activation requires concatemerization ( Furuya et al . , 2017; Kawano et al . , 2015 ) , which may affect trafficking , motility and function of target proteins , create vector payload constraints , and give rise to recombination and/or silencing of the sequence repeats . Here , we overcome these limitations of the Magnets by structure-guided protein engineering and validation by cellular assays . The resulting reagents , ‘enhanced Magnets’ ( eMags ) , have greater thermal stability and dimerization efficiency , as well as faster association and dissociation kinetics . We confirmed their effectiveness in a variety of applications including protein recruitment to different organelles , the generation/expansion of organelle contact sites , and the rapid and reversible reconstitution of VAP-dependent inter-organelle tethers that have key regulatory functions in lipid transport .
Optimal photo-heterodimerizer performance convolves together several parameters: ( i ) Efficient , fast interaction of the two different components upon light stimulus , ( ii ) little or no formation of homodimers – which would compete with productive heterodimer complexes , ( iii ) low background before light stimulus; and ideally , ( iv ) fast heterodimer dissociation following light offset . The existing Magnets systems , especially the Fast1 and Fast2 variants with fast dissociation kinetics ( Kawano et al . , 2015 ) , have weak dimerization efficiency and thus perform poorly on the first criterion , necessitating the use of concatemers ( usually three copies ) of either or both monomers to achieve acceptable reconstitution in a number of settings ( Benedetti et al . , 2018; Furuya et al . , 2017; Kawano et al . , 2015 ) . A pair with greater dimerization efficiency would be desirable , ideally allowing single copies of the complementary Magnets to suffice . With the goal of engineering such a pair , we first established a robust screen for reconstitution of Magnets dimerization using light-dependent accumulation of a protein at the outer mitochondrial membrane ( Benedetti et al . , 2018; Figure 1A ) , which is readily visible and quantifiable . The nMagHigh1 monomer , tagged with the green fluorescent protein EGFP , was used as bait on the outer mitochondrial membrane by fusion to the transmembrane C-terminal helix from OMP25 ( ‘nMag-EGFP-Mito’ ) ( Figure 1—figure supplement 1A and Supplementary file 1 ) . The pMagFast2 monomer , tagged with the red fluorescent protein TagRFP-T ( Shaner et al . , 2008 ) , was used as the cytoplasmic prey ( ‘pMag-TagRFP-T’; Figure 1—figure supplement 1A , Supplementary file 2 ) . We co-expressed both constructs in HeLa cells by co-transfection , grew cells at 28°C for 24 hr , and tested light-dependent prey capture and release by the bait ( Figure 1B , Video 1 ) . Short ( 1 min of 200 ms light pulses every 2 s ) irradiation with cyan light ( 488 nm; 3 × 10−3 W/cm2 ) sufficed to recruit the prey from its diffuse cytoplasmic distribution ( Figure 1B , 2nd panel ) to mitochondria ( Figure 1B , 3rd panel ) , resulting in a precise overlapping localization of prey and bait ( Figure 1—figure supplement 2A ) . This recruitment was reversible following light offset ( Figure 1B , 4th panel ) . Importantly , excitation light for TagRFP-T , as well as that for mCherry and the infrared fluorescent protein iRFP ( Shcherbakova and Verkhusha , 2013 ) , is well outside the action spectrum of LOV domain proteins ( 400–500 nm light excitation ) ( Losi et al . , 2018 ) ; EGFP excitation light is coincident with Magnets activation and is thus used sparingly in these experiments . Next , we began the process of Magnets redesign by optimizing the placement of charge-complementing amino acids in the Vivid dimer interface , using the crystal structure of the light-activated dimer ( PDB ID 3RH8 ) ( Vaidya et al . , 2011; Figure 1—figure supplement 3A–C ) as a guide , and mitochondrial recruitment as the testbed . The original Magnets pair was built upon the mutations Ile52 and Met55 to Arg ( positive Magnet ) and Ile52 to Asp and Met55 to Gly ( negative Magnet ) within the Ncap domain ( See Figure 1—figure supplement 3A ) , which mediates dimerization . To achieve more efficient dimerization , we first sought to optimize charge placement at the interface . Substitution of Asp52 to Glu in nMag-Asp52Glu to modify the position of the negative charges somewhat disrupted heterodimerization , consistent with Kawano et al . , 2015 . We next tried to introduce two negative charges into nMag , at the same two sites where positive charges had been introduced into pMag . nMag-Gly55Glu completely inhibited heterodimerization , whereas nMag-Gly55Asp somewhat improved it ( Figure 1—figure supplement 3D ) . Adding a third positive charge to pMag at position 48 also completely disrupted heterodimerization . In the end , we left the charges alone and instead sought to improve heterodimer interface packing and helical preference with nMag-Gly55Ala , which indeed improved both heterodimerization efficiency and association kinetics – more so than nMag-Gly55Asp . In fact , the nMag-Gly55Ala mutation alone sufficiently improved mitochondrial recruitment after preincubation at 28°C so that it functioned well as a monomer ( Figure 1—figure supplement 3D ) . Having improved the system to allow single-copy use at 28°C , we next sought to improve the temperature stability of the proteins to allow experiments at 37°C . As before , recruitment to the mitochondrial membrane in HeLa cells was used as the cellular assay: nMagHigh1-Gly55Ala-EGFP-OMP25 and pMagFast2-TagRFP-T were co-expressed on the outer mitochondrial membrane and in the cytoplasm , respectively , of HeLa cells by co-transfection . Identical amounts of DNA , in the same plasmid ratio , were used , to allow side-by-side quantification of expression level , background association in the dark , heterodimerization efficiency , and kinetics of association and dissociation . Cells were preincubated at 28°C , 33°C , 35°C , or 37°C for 12–24 hr and then imaged at 37°C to quantify mitochondrial accumulation . We made and tested a number of mutants ( Figure 1—figure supplement 3A , Supplementary file 3 ) in the assay . Mutations were designed according to multiple criteria: removal of potential ubiquitination sites , improvement in secondary-structure preference , and mutations based on the homologous Vivid domains of the thermophilic ascomycetes Thielavia terrestris , Myceliophthora thermophila , Chaetomium thermophile , Rhizomucor pusillus , Rhizomucor miehei , Thermomucor indicae , and Thermothelomyces thermophilus ( Figure 1—figure supplement 4 ) , which have optimal growth temperatures around 50°C ( de and Rodrigues , 2019 ) . Mutations were introduced into both nMagHigh1-Gly55Ala and pMagFast2 components . A number of single mutations improved dimerization efficiency and/or kinetics upon preincubations at 28°C and higher temperatures ( Supplementary file 3 ) . Of the individual mutations tested , Thr69Leu , Met179Ile , and Ser99Asn ( all from thermophilic homologues ) each improved dimerization efficiency at 28°C , and the latter allowed it at 33°C . Thr69Leu is in the interface and improves hydrophobic interactions ( Figure 1—figure supplement 5A , B ) , Met179Ile is in the hydrophobic core and improves packing ( Figure 1—figure supplement 5C , D ) , and Ser99Asn is surface-exposed and optimizes hydrogen bonding and secondary-structure preference ( Figure 1—figure supplement 5E , F ) . Combining these three mutations substantially increased dimerization at both 28°C and 33°C , and all further variants were tested on top of this combination . The mutation Val67Ile increased dimerization efficiency at 33°C; however , it also slowed recovery kinetics – thus , we did not include it . Mutations of Asn133 to lysine , phenylalanine , or tyrosine ( the latter two from thermophiles ) both enhanced dimerization at 33°C , with Asn133Phe and Asn133Tyr facilitating it at 35°C , with Asn133Tyr having slightly stronger dimerization but somewhat slower dissociation kinetics than Asn133Phe . The additional Tyr94Glu mutation ( from thermophiles , improves helical preference ) permitted weak dimerization at 37°C with dissociation kinetics comparable to the original Magnets molecules . The adjacent mutations Asn100Arg/Ala101His ( from thermophiles , improve helical preference ) allowed stronger 37°C dimerization . Finally , Arg136Lys ( from thermophiles , improves helical preference , improves electrostatics with FAD cofactor; Figure 1—figure supplement 5G , H ) further increased dimerization efficiency . During our screening we identified several point mutations that completely abolished the functionality of the pair even upon 28°C incubation , for example Gly49Ala , Tyr50Phe or Ile , Asn56Thr , Tyr87Phe , Val103Ile , Arg106Lys , Lys125Arg , Asp128Ala or Glu , Asn130Glu , Ile139Leu , Phe162Ile or Leu , Ser178Cys or Phe ( See Supplementary file 3 for details; other mutations worsened performance without abolishing it ) . These results will help elucidate LOV domain structure-function relationships – particularly in the absence of comprehensive structural characterization of the light-dependent conformational changes in LOV domain dimers . We selected a pair of variants , eMags , with these eight mutations ( Thr69Leu , Tyr94Glu , Ser99Asn , Asn100Arg , Ala101His , Asn133Tyr , Arg136Lys , and Met179Ile ) added to nMagHigh1-Gly55Ala and pMagFast2 . eMags supports dimerization upon growth at 37°C without preincubation at a lower temperature , while the original Magnets variants were completely nonfunctional after these growth conditions ( Figure 1C , D , Figure 1—source data 1 , Figure 1—figure supplement 6 , Figure 1—figure supplement 6—source data 1 ) . eMags show greater dimerization efficiency ( ~4–5 x ) , as judged by greater prey accumulation on mitochondria ( p=0 . 0004 , Kruskal-Wallis and Dunn’s multiple comparison post hoc tests; Figure 1D ) and faster association and dissociation kinetics ( τON = 3 . 6 ± 0 . 3 s , τOFF = 23 . 1 ± 0 . 6 s ) than original Magnets in cells preincubated at 28°C ( τON = 7 . 6 ± 0 . 3 s , τOFF = 32 . 0 ± 1 . 3 s; p<0 . 0001 for both τON and τOFF , unpaired Student’s t-test; Figure 1C ) . The Tyr133Phe mutation in eMags produced eMagsF , with similar but slightly lower dimerization efficiency as eMags , but significantly faster association and dissociation kinetics ( τON = 2 . 8 ± 0 . 3 s , τOFF = 14 . 0 ± 0 . 6 s; p<0 . 0001 for both τON and τOFF , unpaired t-test; Figure 1C ) . A 3x prey concatemer ( i . e . nMagHigh1-EGFP-OMP25 and pMagFast2 ( 3x ) -TagRFP-T ) – still requiring preincubation at 28°C – is needed to bring the prey recruitment of original Magnets in line with that of monomeric eMags and eMagsF ( Figure 1D ) . This concatemerized original Magnets also suffers from slower association and dissociation kinetics ( τON = 5 . 6 ± 0 . 5 s , τOFF = 45 . 9 ± 1 . 4 s; p = <0 . 0001 for both τON and τOFF , unpaired t-test; Figure 1C , D ) . We refer to nMagHigh1-Gly55Ala and pMagFast2 with these eight mutations as eMagA ( Acidic heterodimerization interface ) and eMagB ( Basic heterodimerization interface ) , respectively . We then sought to establish performance of the new eMags constructs in a variety of experimental contexts . In the first , we used eMags to conditionally recruit cytosolic proteins to intracellular organelles other than mitochondria . For the endoplasmic reticulum ( ER ) , we selected the N-terminal transmembrane domain of cytochrome P450 ( Szczesna-Skorupa and Kemper , 2000 ) , which displays on the cytoplasmic face of the ER , as bait ( fused to EGFP ) . Co-expression of this construct , ER-EGFP-eMagA , with eMagB-TagRFP-T ( prey ) in COS7 cells showed large , rapid , reversible accumulation of prey to the ER upon whole-cell illumination ( Figure 2A , Figure 1—figure supplement 2B , and Video 2 ) ( See Figure 1—figure supplement 1A , B , Supplementary file 1 and 2 , Methods for a complete list and detailed information on bait and prey constructs used in these experiments ) . With focal illumination , robust prey accumulation occurred only in the irradiated ER region ( Figure 2B and Video 3 ) , in spite of the known rapid diffusion of proteins within the ER network ( Nehls et al . , 2000 ) . For recruitment to lysosomes , we used the N-terminal transmembrane sequence of Late Endosomal/Lysosomal Adaptor , MAPK and mTOR Activator 1 ( p18/LAMTOR1 ) , the principal lysosomal surface anchor protein for the mTOR pathway ( Nada et al . , 2014 ) . We co-expressed this bait , Lys-eMagA-EGFP , prey eMagB-TagRFP-T , and lysosomal marker Lamp-1-iRFP in primary mouse hippocampal neurons ( 14 DIV ) ; focal illumination of single lysosomes drove prey recruitment selectively to these isolated organelles ( Figure 2C and Video 4 ) , demonstrating the excellent spatial precision of eMags photoactivation . Finally , for recruitment to the plasma membrane ( PM ) , we targeted eMagAF-EGFP bait to the cytoplasmic PM face with the CAAX-box membrane-targeting sequence from N-ras ( Choy et al . , 1999 ) . As prey , we used mCherry-eMagBF for fluorescence visualization , and also fused the catalytic domain from the inositol 5-phosphatase OCRL ( Pirruccello and De Camilli , 2012 ) , which dephosphorylates phosphatidylinositol 4 , 5-bisphosphate ( PI ( 4 , 5 ) P2 ) . To monitor the degradation of PI ( 4 , 5 ) P2 by the recruited OCRL , we expressed a third fluorescent protein , iRFP , fused to the Pleckstrin homology ( PH ) domain of phospholipase-Cδ1 ( PHPLCδ ) , which selectively binds PI ( 4 , 5 ) P2 over other lipid head groups ( Hammond and Balla , 2015 ) and thus serves as a localization sensor for PI ( 4 , 5 ) P2 . All three constructs were co-expressed in primary hippocampal neurons ( 7 DIV ) . Blue-light irradiation of cells induced rapid accumulation of mCherry signal at the PM ( reflecting OCRL recruitment ) and subsequent iRFP signal loss from the PM ( reflecting OCRL activity converting PI ( 4 , 5 ) P2 to phosphatidylinositol 4-phosphate ( PI4P ) and subsequent PHPLCδ release to the cytoplasm; Figure 2D–G and Video 5 ) . iRFP signal rapidly decayed ( τON = 18 . 1 ± 4 . 6 s ) , indicating rapid eMags binding , OCRL activity , and PHPLCδ unbinding . Upon interruption of blue light irradiation , the iRFP signal quickly recovered ( τOFF = 23 . 4 ± 1 . 6 s ) , indicating fast eMags unbinding , PI ( 4 , 5 ) P2 resynthesis , and PHPLCδ binding ( yielding iRFP signal recovery ) ( Figure 2E , Figure 2—source data 1 ) . Importantly , multiple cycles of illumination produced essentially identical waveforms of iRFP signal and recovery to initial levels , showing that both eMags binding and unbinding , and PI ( 4 , 5 ) P2 generation , are readily reversible with little drift from baseline ( Figure 2F ) . In another set of applications , we validated the efficiency of eMags to induce organelle contacts ( Figure 3A , Figure 1—figure supplement 1D ) . Conditional induction or expansion of such contacts may help elucidate the contribution of inter-organelle contacts and signaling to a variety of biochemical pathways . We first designed a light-inducible ER-lysosome tethering system . Using the targeting sequences above ( Figure 2 ) , ER-mCherry-eMagA and Lys-eMagB-iRFP were co-transfected into COS7 cells . Before blue light activation , ER-lysosome overlap , as detected by mCherry and iRFP overlap , was minimal ( Figure 3B ) ; during 1 min . irradiation , overlap rapidly increased by ~50% ( τON = 7 . 5 ± 0 . 8 s , N = 14 cells , three independent experiments , Figure 3—source data 1 ) , most likely through expansion of pre-existing contacts or by stabilization and expansion of new contacts . Following light offset , ER-lysosome overlap declined quickly to baseline ( τOFF = 35 . 9 ± 1 . 7 s; Figure 3B , Video 6 ) . The longer time courses of organelle association-dissociation ( tens of seconds ) , relative to cytoplasmic protein recruitment ( seconds ) , is consistent with a combination of slower mobility of organelles than free protein and the processive assembly and disassembly of membrane contacts . Using a similar targeting strategy , ER-mCherry-eMagA and eMagB-iRFP-Mito were used to drive ER-mitochondrial association ( Figure 3C ) . In HeLa cells , used for these experiments , ER and mitochondria form a closely interacting network even in control conditions . Upon 2 min . irradiation , however , overlap increased by ~20% , with kinetics ( τON = 28 . 0 ± 1 . 9 s , τOFF = 49 . 1 ± 2 . 5 s , N = 14 cells , three independent experiments; Figure 3C , Video 7 , Figure 3—source data 1 ) on the order of that seen for ER-lysosomes contacts . Finally , for mitochondrion-lysosome manipulation , we used eMagA-mCherry-Mito and Lys-eMagB-iRFP . In HeLa cells , baseline overlap was quite low ( Figure 3D ) ; such contacts are typically transient and involve small contact area ( Wong et al . , 2018 ) . Upon activation , increased associations between lysosomes and mitochondria were observed , revealing contact expansion ( τON = 40 . 1 ± 2 . 6 s , τOFF = 58 . 4 ± 2 . 6 s , N = 17 cells , two independent experiments; Video 8 , Figure 3—source data 1 ) . In some cases , movement of lysosomes away from mitochondria resulted in the elongation of tubules from mitochondria , and even in their fission ( Figure 3E , Video 9 ) , indicating strong association . In a final application , we tested eMags for acute manipulation of intracellular PI4P via reconstitution of an ER-transGolgi network ( TGN ) tether . Key components of this tether are the ER protein VAMP-associated protein ( VAP ) and Oxysterol-binding protein 1 ( OSBP1 ) . OSBP1 , which binds VAP ( via an FFAT motif ) and membranes of the TGN ( via a PI4P-binding PH domain ) , also contains an ORD domain ( OSBP-related domain ) that promotes exchange of TGN PI4P for ER cholesterol ( Murphy and Levine , 2016 ) . Following shuttling to the ER , PI4P is degraded by the phosphatidylinositide phosphatase Sac1 ( Mesmin et al . , 2013; de Saint-Jean et al . , 2011; Zewe et al . , 2018 ) . This model of ER-Golgi PI4P transport is supported by biochemical , pharmacological , and genetic studies ( Dong et al . , 2016; Mesmin et al . , 2013; Strating et al . , 2015 ) . We sought to use the eMags tools to offer direct optogenetic control over this PI4P-cholesterol exchange through regulation of VAP-OSBP1 binding interactions . The overall design strategy was to replace endogenous VAP with a split version , which could be reconstituted by eMags dimerization and would then associate with OSBP1 to drive transport . Unlike the earlier examples , this necessitated careful consideration of the domain architectures of VAP and OSBP1 , to best ensure that ( 1 ) split-VAP would not reconstitute in the absence of light activation and ( 2 ) that the eMagA and eMagB fusions would not interfere with either VAP reconstitution or OSBP1 interaction . VAP is an integral membrane protein composed of a cytosolic major sperm protein ( MSP ) domain ( which binds FFAT motif-containing proteins ) , a coiled-coil domain and a C-terminal membrane anchor ( Kaiser et al . , 2005; Kim et al . , 2010; Figure 4A and Figure 1—figure supplement 1E ) . Two distinct VAP genes exist in the vertebrate genome: VAPA and VAPB , which can form either homomers or heteromers with one another . OSBP1 has an N-terminal PH domain that preferentially binds PI4P ( Mesmin et al . , 2013; Murphy and Levine , 2016; Venditti et al . , 2019 ) , an internal FFAT motif , and a C-terminal ORD domain which binds in a competitive way PI4P and cholesterol . Given this domain structure , we opted to convert VAPB into a cytosolic version through deletion of the C-terminal transmembrane helix ( leaving VAPB ( 1-218 ) ) ; we retained the MSP and coiled-coil domains as both may contribute to VAP dimerization ( Kim et al . , 2010; Figure 4B ) . We fused TagRFP-T to the N-terminus of this cytosolic fragment , and eMagB to its C-terminus ( TagRFP-T-VAPB ( 1-218 ) - eMagB; Figure 4B , Figure 1—figure supplement 1E and Supplementary file 2 ) . We then used ER-eMagA-EGFP to recruit VAPB ( 1-218 ) to the ER upon blue light irradiation , where it could interact with OSBP1 . We refer to this pair of constructs as ‘Opto-VAP’ . We first tested the efficiency of Opto-VAP by transfecting both components into HeLa cells and imaging them by confocal microscopy . The prey protein ( TagRFP-T-VAPB ( 1-218 ) -eMagB ) was imaged throughout the experiment , while ER-eMagA-EGFP was imaged only during optogenetic activation . Before blue light irradiation , the prey protein was homogeneously distributed throughout the cytosol , with focal accumulation around the Golgi ( Figure 4C ) . We interpret this observation as reflecting interaction of VAPB with endogenous OSBP1 , which is abundant in the Golgi , where it binds the PI4P-rich TGN membranes via its PH domain ( Mesmin et al . , 2013 ) . The cytosolic VAPB ( 1-218 ) prey , with its MSP domain , could compete with endogenous VAP for binding to the FFAT motif of OSBP1 ( Figure 4A , B ) . A robust presence of PI4P in the TGN under resting conditions was confirmed by strong colocalization with co-transfected PI4P reporter iRFP-P4C ( Hammond and Balla , 2015; Luo et al . , 2015; Figure 4C ) . Upon irradiation with blue light ( 50 ms blue-light pulses at 0 . 5 Hz for ~1 min . ) , there was a massive recruitment of TagRFP-T-VAPB ( 1-218 ) -eMagB to the ER ( Figure 4C -top , Figure 4—figure supplements 1 and 2A ) , consistent with VAP-OSBP1-based reconstitution of ER-TGN interactions . Concomitant with this was a rapid ( τON = 22 . 8 ± 2 . 4 s ) reduction of iRFP fluorescence in the Golgi ( Figure 4C -bottom , Video 10 , Figure 4—source data 1 ) , approaching a plateau of ~70% of resting in approximately 50 s . This suggests that optogenetic reconstitution of split-VAP indeed restores a VAP-OSBP1-dependent ER-TGN tether and resulting transport of PI4P from the Golgi to the ER . These changes were rapidly reversed after interruption of blue light , with the full regeneration of the PI4P signal to baseline occurring in approximately 5 min ( τOFF = 143 . 8 ± 3 . 8 s ) . To confirm that the observed PI4P transfer was indeed mediated by OSBP and Opto-VAP , cells were preincubated for 30 min with 10 μM itraconazole ( ITZ ) , an antifungal and anticancer agent that occludes the lipid-transport domain of OSBP and thus blocks its lipid trafficking properties ( Strating et al . , 2015 ) . After ITZ treatment , no change was detected in the accumulation of the PI4P probe ( iRFP-P4C ) at the Golgi ( graph in Figure 4C and Figure 4—figure supplements 1 and 2A , Figure 4—source data 1 , Figure 4—figure supplement 2—source data 1 ) , despite the efficient recruitment of TagRFP-T-VAPB ( 1-218 ) -eMagB to the ER membrane ( N = 16 cells , two independent experiments ) . We next tested the Opto-VAP system in gene-edited HeLa cells lacking both VAP genes ( VAP double-KO cells ) . It was reported that in these cells the Golgi complex is partially disrupted , with formation of PI4P-enriched hybrid Golgi-endosome structures ( Dong et al . , 2016 ) , a finding that we have confirmed in cells kept in the dark ( Figure 4D -bottom ) . Blue light activation led to rapid recruitment of TagRFP-T-VAPB ( 1-218 ) -eMagB to the ER ( Figure 4—figure supplement 2B ) , whose reticular appearance was less obvious in these cells ( Figure 4D -bait panel ) due to their greater thickness relative to the COS7 cells used in other experiments . Concomitant with VAPB ( 1-218 ) recruitment to the ER , rapid ( τON = 15 . 7 ± 1 . 2 s ) decrease in iRFP fluorescence from the Golgi and hybrid Golgi-endosome structures was observed ( Figure 4D , Figure 4—source data 1 and Video 11 ) , indicating PI4P loss . Thus , Opto-VAP is able to fully restore the activity of the deleted VAPA and VAPB genes in recruiting OSBP1 to perform PI4P-cholesterol exchange . After blue-light interruption , both Opto-VAP localization and PI4P levels reverted to baseline ( τOFF = 93 . 7 ± 5 . 0 s ) ( Figure 4D ) ( N = 20 cells , four independent experiments ) . As before , ITZ completely inhibited PI4P transport but had no effect on Opto-VAP recruitment ( N = 16 cells , three independent experiments ) ( Figure 4D , Figure 4—source data 1 , Figure 4—figure supplement 2B , Figure 4—figure supplement 2—source data 1 ) . The time courses of Opto-VAP recruitment and recovery , and of PI4P loss and recovery , are similar between the wild-type and double-KO cells , suggesting that Opto-VAP assembly and function are largely independent of endogenous levels of VAPA and VAPB . As a final verification of the necessity of the ORD domain in the observed PI4P transport , we constructed TagRFP-T-eMagB-PHOSBP , with the PH domain of OSBP1 but not the ORD domain ( Figure 4A , Figure 1—figure supplement 1E , Figure 4—figure supplement 3A and Supplementary file 2 ) . In both wild-type and VAP-DKO HeLa cells , blue-light activation induced rapid prey recruitment to the ER , but with no accompanying changes in iRFP-P4C fluorescence ( Figure 4—figure supplement 3B , C and Figure 4—figure supplement 3—source data 1; n = 16 cells for HeLa , n = 17 for VAP-DKO , two independent experiments ) . Thus , the ORD domain is critical for PI4P transport , with the PH domain alone having no effect .
In this work , we have both engineered a dramatically improved photodimerizer pair and used it in a set of experiments elucidating details of organellar interactions and cellular lipid metabolism and transport . In a previous study ( Benedetti et al . , 2018 ) , we had compared multiple optogenetic dimerizer reagents and found that the Magnets system , based on orthogonalization of the Vivid LOV domain homodimer ( Kawano et al . , 2015 ) , offers major advantages over other systems in several different assays . Magnets have rapid association and dissociation kinetics and require both monomers to undergo blue-light activation to permit dimerization . These properties make the background activation of Magnets low , so that they are well-suited to optogenetic modulation of small volumes and sub-cellular organelles . However , the existing Magnets tools have two critical disadvantages , which preclude their wider adoption: ( 1 ) their weak dimerization efficiency necessitates the use of concatemers , which can perturb target proteins and slow kinetics , and ( 2 ) the low thermodynamic stability means that expression and maturation must occur at reduced temperatures , complicating cell-culture experiments and ruling out mammalian in vivo work entirely . To overcome these limitations , we established a robust cell-culture screen that captures dimerization efficiency , association and dissociation kinetics , and folding and maturation . This screen allowed us to identify variants encompassing mutations across the whole protein with particular focus on the dimer interface . Mutations were selected based on sequence alignments with thermophilic fungal Vivid domains and structure-guided design . After several rounds of mutagenesis and screening , we selected final ‘enhanced Magnets’ ( eMag ) variants with nine mutations over the starting scaffolds . The eMag reagents showed greater dimerization efficiency – allowing use as monomers instead of concatemers , full function after their folding and maturation at 37°C , and faster association and dissociation kinetics than the original Magnets . We recently compared ( Benedetti et al . , 2018 ) the original Magnets system to Cry2/CIB1 ( Kennedy et al . , 2010 ) and iLIDs ( Guntas et al . , 2015 ) , using several of the assays also used in this study , for example protein recruitment to ER , mitochondria , or single lysosomes , and activity of OCRL phosphatase recruited to the plasma membrane . From this systematic comparison , several obvious themes emerged . Firstly , upon prolonged local ( 3 x 3 μm ) illumination , Magnets dimerization occurs and persists only in the irradiated region , while activated iLID dimers extend some distance from this region and activated Cry2/CIB1 dimers penetrate the entire cell . Secondly , recovery kinetics of Cry2/CIB1 after light offset are more than an order of magnitude slower than those of iLIDs and Magnets . Thirdly , the efficiency of light-driven recruitment of original Magnets is greater than that of iLIDs but somewhat lower than that of Cry2/CIB1 in whole cell illumination conditions . Importantly , use of Cry2/CIB1 involves a balance between the desired Cry2/CIB1 heterodimerization and counterproductive Cry2 oligomerization ( Bugaj et al . , 2013; Che et al . , 2015; Duan et al . , 2017; Taslimi et al . , 2016 ) . This balance is difficult to determine and could vary across cellular environments and target proteins . Finally , Cry2 is less effective when used in membrane-bound bait ( Benedetti et al . , 2018; Che et al . , 2015; Hallett et al . , 2016; Pathak et al . , 2014 ) , hampering its utility in applications such as those shown here ( Cry2 is much larger than LOV domains and may perturb fused proteins and organelles ) . Relative to the original Magnets – which we systematically compared to Cry2/CIB1 and iLIDs in our previous study – eMags shows significant improvements in the dimerization efficiency , and association and dissociation kinetics . Taken together , eMags provides several advantages over Cry2/CIB1 and iLIDs for subcellular optogenetics and perhaps other settings as well . We have shown the benefits of the faster eMagsF clones for rapid manipulation . In the other direction , it is likely that grafting the photoactivation-extending mutations Val74Ile and/or Val85Ile ( Zoltowski et al . , 2009 ) could produce high-efficiency , thermostable versions of eMags with recovery kinetics on the order of minutes to hours , if experimenters desire bistable control of protein-protein interactions . We thoroughly validated the eMags constructs in a range of cellular assays both in whole cell and local irradiation conditions involving protein recruitment to different membranes , inter-organellar association , and bilayer lipid metabolism and trafficking . The success of the engineering effort validates the design strategy and shows that many mutations from thermophilic fungi grafted well to the scaffold of the Vivid photoreceptor of Neurospora crassa , a mesophilic fungus . These mutations improved packing , hydrogen bonding , and secondary-structure preference . These improved optogenetic dimerizers will be broadly applicable and useful for applications across diverse fields . Also , our protein design and cellular screening strategies will likely extend to other reagent optimization projects .
Expression vectors encoding nMagHigh1-EGFP-CAAX , pMagFast2 ( 3x ) -iRFP and iSH2-pMag ( 3x ) -iRFP were kind gifts from Moritoshi Sato ( University of Tokyo , Tokyo , Japan ) . nMagHigh1-EGFP-Mito was generated through the PCR amplification of the nMagHigh1-EGFP coding sequence from nMagHigh1-EGFP-CAAX and inserted into a pGFP-OMP25 ( Nemoto and De Camilli , 1999 ) vector at NheI and XhoI sites . pMagFast2 ( 1x ) -TagRFP-T was generated through the PCR amplification of the third unit of pMagFast2 ( 3x ) and TagRFP-T in pMagFast2 ( 3x ) -TagRFP-T ( Benedetti et al . , 2018 ) and inserted in the same vector at HindIII and XbaI site . In order to recreate an optimal Kozak sequence Met and Gly were added before the initial His , at the N-term of pMagFast2 in this construct . All nMagHigh1 and pMagFast2 mutants tested in our screening were generated by site-directed mutagenesis ( QuikChange II XL , Agilent technologies ) following manufacturer instruction . The complete list of primers can be found in Supplementary files 4 , 5 . The sequences of the enhanced Magnets mutants generated have been deposited in GenBank: eMagAF ( GenBank accession number: MW203024 ) , eMagBF ( GenBank accession number: MW203025 ) , eMagA ( GenBank accession number: MW203026 ) , eMagB ( GenBank accession number: MW203027 ) . ER-EGFP-eMagA , ER-mCherry-eMagA , eMagA-mCherry-Mito , eMagB-iRFP-Mito , Lys-eMagB-iRFP and TagRFP-T-VAPB ( 1-218 ) -eMagB were generated by GeneScript and cloned into M18 pCAGGS WPRE electroporation vector ( Gray et al . , 2006 ) . These constructs are described in Supplementary file 1 and 2 . Lys-eMagA-EGFP was generated replacing nMagHigh1 with eMagA in Lys-nMagHigh1-EGFP ( Benedetti et al . , 2018 ) . Lamp1-iRFP was generated by PCR-amplifying the Homo sapiens lysosomal associated membrane protein 1 ( LAMP1 ) coding sequence ( NCBI Reference Sequence: NM_005561 . 3 ) synthesized as a gBlocks Gene Fragment ( Integrated DNA Technologies , IDT ) , which was inserted at EcoRI and BamHI sites of piRFP670-N1 ( Addgene plasmid # 45457 ) . eMagAF-EGFP-PM was generated replacing nMagHigh1 in nMagHigh1-EGFP-CAAX with the engineered variant at HindIII and XbaI sites . mCherry-eMagBF−5ptaseOCRL was synthesized by digesting mCherry-pMagFast2 ( 3x ) −5ptaseOCRL ( Benedetti et al . , 2018 ) with NotI and PvuI , and then ligated with eMagBF amplified from eMagBF-TagRFP-T . iRFP-PHPLCδ plasmid was previously described ( Idevall-Hagren et al . , 2012 ) . TagRFP-T-eMagB-PHOSBP coding sequence was synthesized as a gBlocks Gene Fragment ( Integrated DNA Technologies , IDT ) and cloned into TagRFP-T-VAPB ( 1-218 ) -eMagB vector at NotI and HpaI cloning sites . PHOSBP sequence was obtained from GFP-PHOSBP ( Tim Levine , UCL Institute of Ophthalmology ) . iRFP-P4C was cloned amplifying the iRFP coding sequence piRFP670-N1 ( Addgene plasmid # 45457 ) and inserted at AgeI and BsrGI cloning sites in GFP-P4CSidC ( kind gift of Yuxin Mao , Cornell ) . For all of these clones , PCR amplification of the fragments , and their subsequent ligation , was performed using the In-Fusion Cloning Kit and online tools ( BD Clontech , Takara Bio , USA ) . All plasmids were verified by sequencing ( Genewiz , South Plainfield , NJ , USA ) . Thermophilic Vivid domain homologues were found using tblastn of the Neurospora crassa sequence against whole-genome sequences of thermophilic fungi whose identities were found from literature search . Only the closest homologue from each genome was selected for analysis . Several sequences were incomplete , for example the two Rhizomucor homologues . The homologues showed a high level of identity and similarity to the Neurospora crassa sequence , with a number of positions that clustered amongst some or all of the thermophilic sequences , but were different from the Neurospora crassa sequence ( Figure 1—figure supplement 4 ) . These were preliminary considered to be promising sites for mutagenesis . Structural analysis was performed on the 3RH8 PDB file ( annotated as the ‘light-state dimer’ of Vivid ) overlaid with that of 2PD7 ( ‘dark-state monomer’ ) – structures were examined , and images made , with PyMOL 2 . 3 . 5 . Primary attention was paid to the dimer interface , the FAD binding site , and surrounding regions . Potentially stabilizing mutations were selected from side-chains that would improve secondary-structure preference ( e . g . https://bmrb . io/referenc/choufas . shtml ) , hydrophobic exposure , backbone stabilization , or packing – or from side-chains represented at the corresponding position of the thermophilic sequences . Often these were one and the same . Judgments about packing , exposure , backbone stabilization , and clashes were made using the ‘Mutagenesis’ functionality in PyMOL and analysis with the MolProbity server ( http://molprobity . biochem . duke . edu ) . Ubiquitination was predicted using http://bdmpub . biocuckoo . org/prediction . php . Prioritization of mutations to combine was done considering both proximity in space and in linear sequence ( i . e . ability to be encoded on a single primer ) . Wild-type ( ATCC CCL-2 ) and VAP double KO ( DKO ) ( Dong et al . , 2016 ) HeLa cells , as well as COS7 ( ATCC CRL-1651 ) cells , were cultured at 37°C ( 5% CO2 ) in phenol red-free Dulbecco’s Modified Eagle Medium ( DMEM , Life Technologies ) , supplemented with 10% fetal bovine serum ( Life Technologies ) , 1 mM sodium pyruvate ( Life Technologies ) , 100 U/ml penicillin , 100 μg/ml streptomycin ( Life Technologies ) , MEM-Non-Essential Amino Acids ( Life Technologies ) , and 1 mM L-glutamine ( Life Technologies ) . All lines were tested monthly and verified as being mycoplasma-free ( MycoSensor PCR Assay Kit , Agilent Technologies ) . Primary cultures of hippocampal neurons were generated from mouse brains . Hippocampi of P0-P2 C57BL/6 ( Charles River ) pups were dissected in cold Hank’s Balanced Salt Solution [HBSS 1x supplemented with 10 mM HEPES pH 7 . 4 , 100 U/ml penicillin , 100 μg/ml streptomycin , and 1 mM sodium pyruvate ( all reagents from Life Technologies ) ] . Cells were then dissociated by tissue trituration and papain treatment [20 U/ml papain ( Worthington Biochemical corporation ) , and 0 . 2 mg/ml L-cysteine ( Sigma ) in HBSS , pH 7 . 4] at 37°C for 15 min . Live , dissociated cells ( Trypan Blue exclusion ) were counted and seeded in plating medium [Neurobasal supplemented with 5% fetal bovine serum , 1% Glutamax , and 2% B27 ( all reagents from Life Technologies ) ] at 3 . 4 × 104 cells/cm2 on poly-D-lysine-coated ( Sigma ) , glass-bottomed Petri dishes ( MatTek corporation ) . Three hours after transfection , the serum-based medium was replaced with serum-free neuronal medium , and cells were maintained in vitro at 37°C and 5% CO2 . Transient transfection was performed between 4 and 14 days in vitro ( DIV ) . All experimental procedures involving the use of mice were performed in agreement with the Yale University Institutional Animal Care and Use Committee ( IACUC ) ( protocol number 2018–07422 ) , and with the Janelia Farm Research Campus Institutional Animal Care and Use Committee and Institutional Biosafety Committee ( protocol number 18–173 ) . For live-cell imaging experiments , cells were seeded on glass-bottomed dishes ( MatTek corporation ) coated with 0 . 005 mg/ml human plasma fibronectin ( EMD Millipore ) at 37°C for 30 min , and then washed three times with sterile water . Fibroblastic cells were seeded at a concentration of 10–15 × 104 cells/cm2 per dish and transfected after 24 hr with Lipofectamine 2000 ( Life Technologies ) , following the manufacturer’s instructions . To study prey-protein recruitment at mitochondria during mutant screening , cells were transfected with cDNAs encoding nMagHigh1-EGFP-Mito ( bait ) and pMagFast2-TagRFP-T ( prey ) variants at a 1:1 ratio in OptiMEM-I ( Thermo Fisher Scientific ) ( 1:4 DNA: lipofectamine ratio ) . Cells were incubated with the transfection mix for 1 hr . Subsequently , the serum-free medium was replaced by complete DMEM , and cells were incubated at 28 , 33 , 35 , or 37°C for 12–24 hr before imaging . All imaging experiments were performed at 37°C in Live-cell imaging solution ( Life Technologies ) . Single-lysosome prey recruitment was performed in 14 DIV hippocampal neurons transfected with Lys-eMagA-EGFP ( bait ) , eMagB-TagRFP-T ( prey ) , and Lamp1-iRFP ( reporter ) at a 3:2:1 ratio , with 1 . 5 μg total DNA ( 1:4 DNA: lipofectamine ratio ) . Plasma membrane modulation of PI ( 4 , 5 ) P2 was tested in 7 DIV hippocampal neurons transfected with eMagAF-EGFP-PM ( bait ) , mCherry-eMagBF-5ptaseOCRL ( prey ) , and iRFP-PHPLCδ ( reporter ) at a 3:2:1 ratio , with 1 . 5 μg total DNA ( 1:4 DNA: lipofectamine ratio ) . To study prey-protein recruitment at the ER , cells were transfected with cDNAs encoding ER-eMagA ( bait ) and eMagB-TagRFP-T ( prey ) at a 2:1 ratio in OptiMEM-I ( Thermo Fisher Scientific ) ( 1:4 DNA: lipofectamine ratio ) . Light-dependent VAPB reconstitution on ER membranes or PHOSBP-mediated tethering was performed by transfecting wild-type or VAP-DKO HeLa cells with ER-EGFP-eMagA ( bait ) and TagRFP-T-VAPB ( 1-218 ) - eMagB or TagRFP-T-eMagB-PHOSBP ( prey ) and iRFP-P4C at a 3:2:1 ratio in OptiMEM-I ( Thermo Fisher Scientific ) ( 1:4 DNA: lipofectamine ratio ) . In this case , cells were incubated with the transfection mix for 1 hr . Subsequently , the serum-free medium was replaced by complete DMEM with no phenol red , and imaging was performed in the same medium between 16 and 28 hr after transfection . Itraconazole ( Tocris , Cat . No . 5981 ) was dissolved in DMSO to generate a 2 mM solution right before the experiment and diluted in the cell medium at 10 μM final concentration 30 min before imaging in a dark room . Light-dependent induction of contacts between ER and lysosomes was achieved transfecting COS7 cells with ER-mCherry-eMagA and Lys-eMagB-iRFP at a 2:1 ratio in OptiMEM-I ( 1:4 DNA: lipofectamine ratio ) . ER-mitochondria contacts were elicited in HeLa cells transfected with ER-mCherry-eMagA and eMagB-iRFP-Mito at a 1:2 ratio in OptiMEM-I ( 1:4 DNA: lipofectamine ratio ) . Mitochondria-lysosome contacts were evoked in HeLa cells transfected with eMagA-mCherry-Mito and Lys-eMagB-iRFP at a 2:1 ratio . Cells were incubated with the transfection mix for 1 hr . Subsequently , the serum-free medium was replaced by complete DMEM with no phenol red , and imaging was performed in the same medium between 16 and 28 hr after transfection . All optogenetic experiments , with the exception of the experiments with Opto-VAP and its controls and the light-dependent induction of inter-organellar contacts , were performed using the Improvision UltraVIEW VoX system ( Perkin Elmer ) , built around a Nikon Ti-E inverted microscope and controlled by the Volocity software ( Improvision ) . Imaging was carried out at 37°C with a 63x PlanApo oil objective ( 1 . 45 NA ) . To prevent unwanted photoactivation of the optogenetic dimerizers , transfected cells were identified with the fluorescence emitted by red fluorescent proteins using a bandpass excitation filter ET 560/30 . A 488 nm laser was used to excite EGFP , a 561 nm laser for mCherry and TagRFP-T , and a 640 nm laser for iRFP670 . The fluorescence emitted was detected with 527/55 nm , 615/70 nm and 705/90 nm filters , respectively . Whole-cell activation of the photoswitches was achieved by irradiating the field of view with 488 nm laser pulses of 100–200 ms ( 3 × 10−3 W/cm2 ) . A built-in photo-perturbation unit was used to deliver 488 nm light ( 7 . 07 W/cm2 ) pulses with subcellular precision . Confocal imaging of light-dependent VAPB reconstitution on ER membranes or PHOSBP mediated tethering and light-induced inter-organellar contacts was performed using a customized Nikon Ti-E inverted microscope outfitted with a Yokagowa CSU-X1 spinning disk . Illumination was generated using solid-state laser lines at 488 nm , 561 nm , or 647 nm passed through the pinhole array and into the back aperture of the objective using a quad-pass filter for the appropriate lines ( Semrock ) . Emission light was collected using a 100x Plan-Apochromat 1 . 49NA oil-immersion objective ( Nikon ) and focused on a DU-897 EMCCD ( Andor ) at a final pixel size of 133 . 3 nm . Specific settings for each color were as follows: 488 – 525/50 emission filter , 50 ms exposure time; 561—605/55 emission filter , 200 ms exposure time; 647—700/75 emission filter , 200 ms exposure time . Cells were imaged in DMEM without phenol red and incubated using a TokaiHit stage-top incubator at 37°C , 5% CO2 . Association and dissociation rates for each dimerization system were calculated from changes in prey fluorescence inside a cytosolic ROI before , during , and after the photoactivation and recruitment of the prey protein to mitochondrial membranes . The change in average fluorescence inside the ROI was calculated using the software Fiji ( ImageJ Version: 2 . 0 . 0-rc-69/1 . 52 p , Wayne Rasband , National Institute of Health , USA , http://fiji . sc/wiki/index . php/Fiji ) , and the remainder of the quantification was carried out in MATLAB . The change in fluorescence associated with depletion of the cytosolic pool was calculated as Δff∘= fti-f[t∘]f[t∘] , where ft= ( FROI[t]-Fbkg[t] ) / ( Fcell[t]-Fbkg[t] ) , where FROI is the mean fluorescence measured in the cytosolic ROI , Fbkg is the mean fluorescence intensity measured in an area of the background , Fcell is the mean fluorescence measured in the whole cell to normalize for photobleaching , and ti denotes the point in time . Changes in iRFP-PHPLCδ at the plasma membrane in neurons was calculated with the same equation but in this case , the region of interest for each time point was identified by manually drawing an ROI corresponding to the plasma membrane . The fluorescence changes due to protein recruitment to mitochondria were calculated by measuring the fluorescence signal corresponding to mitochondria at each time point by generating a binary mask using the fluorescence signal associated with the mitochondrial bait . Fluorescence accumulation at mitochondria was measured by dividing the average background-subtracted fluorescence intensity at every time-point ( Ft ) by the fluorescence intensity of the first time point ( F0 ) and subsequently normalized to ( Ft-F0 ) /F0 . The relative increase in organelle overlap for each time point upon light-dependent induction of membrane contact sites was performed by generating a binary mask using the fluorescence signal associated with lysosomes in ER-lysosome and mitochondria-lysosome contacts , or with mitochondria in ER-mitochondria contacts . Then the fluorescent signal of the other organelle , corrected for background signal and photobleaching-corrected with the Bleach Correction function in Fiji , was calculated with the following equation ft= ( FROI[t]-Fbkg[t] ) and normalized to the fluorescence value measured at the beginning of the experiment . To measure loss of PI4P from the Golgi complex and/or Golgi-endosome-hybrid organelles in experiments involving Opto-VAP , or PHOSBP mediated tethering , in iRFP-P4C expressing HeLa cells , an ROI was drawn around Golgi marker-positive regions . The fluorescence in the ROI at each time point was background-subtracted and photobleaching-corrected using this equation ft= ( FROI[t]-Fbkg[t] ) / ( Fnucleus[t]-Fbkg[t] ) . TagRFP-T-MSPVAPB-eMagB recruitment to the ER was calculated by measuring the fluorescence signal corresponding to the ER at each time point by generating a binary mask using the fluorescence signal associated with the ER bait acquired during the blue-light stimulation of the optogenetic system . Fluorescence accumulation at the ER was measured dividing the average fluorescence intensity at every time-point ( Ft ) background subtracted ( Fbkg ) by the fluorescence intensity of the first time point ( F0 ) background subtracted according to the formula: ( Ft-Fbkg ) / ( F0-Fbkg ) . Statistical analyses were carried out in GraphPad Prism 8 . 2 . 1 ( Graph Pad Software ) . We found that the apparent kinetics of the Magnets variants reported in this study fit well to an exponential decay model . We used the curve-fitting tool ( cftool ) in MATLAB to determine the kinetic rate constants , τON and τOFF , by fitting the curve to the following equation:St=S0+∆S e-t-t0τ Where S=Δff∘ , t0 is time at which the light is turned on or off ( for on- or off-kinetics , respectively ) , S0 is S at time t0 , and ∆S=S0-S ( ∞ ) . During the fitting process , each point is given a weight proportional to 1s . e . m . 2 . The parameters of the fit can be found in Supplementary file 6 . For all the datasets acquired in this work , the R2’s obtained for exponential fits are always larger than 0 . 86 with a median of 0 . 98 .
Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion . Among them , Magnets are small modules engineered from the Neurospora crassa photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers . Both Magnets components , which are well-tolerated as protein fusion partners , are photoreceptors requiring simultaneous photoactivation to interact , enabling high spatiotemporal confinement of dimerization with a single excitation wavelength . However , Magnets require concatemerization for efficient responses and cell preincubation at 28°C to be functional . Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation . We validated these ‘enhanced’ Magnets ( eMags ) by using them to rapidly and reversibly recruit proteins to subcellular organelles , to induce organelle contacts , and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism . eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes .
The cell relies on direct interactions among proteins and compartments called organelles to stay alive . Manipulating these interactions allows researchers to control a wide variety of cell behaviors . A system called ‘Magnets’ uses light to trigger interactions between proteins . Magnets uses a segment of a protein called Vivid from a common bread mold that responds to light . When light shines on two of these segments , it causes them to bind together , in a process known as dimerization . In the Magnets system , Vivid segments are attached to specific proteins or organelles . By using light , researchers can force their target molecules to come together and trigger signals that can change cell behavior . However , the Magnets system has limitations: its stability and low efficiency mean that the cells need to be kept at low temperatures and that several copies of Vivid are needed . These conditions can interfere with the activity of the target proteins . To expand the technique , Benedetti et al . added mutations to make the Vivid protein more similar to proteins found in fungi that thrive at temperatures around 50°C . These changes meant that the enhanced system could work at body temperature in mammals . Further mutations at the interface between the two Vivid segments improved the efficiency of dimerization . This enhanced version was put to the test in different applications , including delivering proteins to different organelles and bringing organelles together . The enhanced Magnets system should enable researchers to control a greater variety of signaling events in the cell . In addition , the methodology established for improving the efficiency of the Magnets system could be useful to researchers working on other proteins .