A large number of GEPP with varied 1O2 quantum yields have appeared recently; therefore, in the present work, the efficacy of different GEPP to photodynamically activate CCK1R was examined, as monitored by Fura-2 calcium imaging. KillerRed, miniSOG, miniSOG2, singlet oxygen protein photosensitizer (SOPP), flavin-binding fluorescent protein from Methylobacterium radiotolerans with point mutation C71G (Mr4511C71G), and flavin-binding fluorescent protein from Dinoroseobacter shibae (DsFbFP) were expressed at the plasma membrane (PM) in AR4-2J cells, which express endogenous CCK1R. Light irradiation  of GEPPPM-expressing AR4-2J was found to all trigger persistent calcium oscillations, a hallmark of permanent photodynamic CCK1R activation; DsFbFP was the least effective, due to poor expression. miniSOG was targeted to PM, mitochondria (MT) or lysosomes (LS) in AR4-2J in parallel experiments; LED light irradiation was found to all induce persistent calcium oscillations. In miniSOGPM-AR4-2J cells, light emitting diode (LED) light irradiation-induced calcium oscillations were readily inhibited by CCK1R antagonist devazepide 2 nM; miniSOGMT-AR4-2J cells were less susceptible, but miniSOGLS-AR4-2J cells were not inhibited. In conclusion, different GEPPPM could all photodynamically activate CCK1R. Intracellular GEPP photodynamic action may prove particularly suited to study intracellular GPCR. Cholecystokinin 1 receptor (CCK1R) is activated by singlet oxygen ( Cholecystokinin 1 receptor (CCK1R) is expressed prominently in highly restricted brain regions, such as the basal ganglia, hippocampus, thalamus, hypothalamus, medulla oblongata ,6,7,8,9,1O2 or 1O2), usually generated in type II photodynamic action with chemical photosensitizer sulphonated aluminum phthalocyanine (SALPC) after a brief cellular incubation [CCK1R is unique among A class G protein-coupled receptors (GPCR) in that it is permanently activated ligand-independently by the lowest lying excited state molecular oxygen, the delta singlet oxygen . Energy transfer to ground state molecular oxygen results in the production of 1O2 (type II) [1O2 quantum yield (ϕ1O2) in type II photodynamic action. In contrast to chemical photosensitizers such as porphyrins and phthalocyanines, genetic manipulations will ensure that GEPP could be targeted to specified cell types or subcellular organelles with high precision. A typical photodynamic action involves three elements: light, a light-absorbing molecule (photosensitizer), and molecular oxygen. After absorption of a photon of a certain wavelength by a photosensitizer, the excited state photosensitizer molecule eventually undergoes either electron transfer or the energy transfer process. Electron transfer leads to the production of oxygen radicals (type I), such as superoxide anion (Otype II) ,30,31. T2−.) [1O2 also [1O2 quantum yield (ϕΔ1O2 0.008) of more than eight-fold higher than O2−. [KillerRed is the first major GEPP to emerge, initially thought to generate solely superoxide anion (O2−.) ,34,35 bu1O2 also ,36, and han O2−. . The minhan O2−. ,39 but hhan O2−. ,41, moduhan O2−. , and forhan O2−. . 1O2 quantum yields  [Q103V) [1O2 quantum yield [1O2-generating flavin-binding fluorescent protein (FbFP) photosensitizers originally from other source organisms, such as Pp2FbFP (from Pseudomonas putida), DsFbFp (from Dinoroseobacter shibae), EcFbFP (from Bacillus subtilis), CreiLOV (from Chlamydomonas reinhardtii), Mr4511C71G (from Methylobacterium radiotolerans), and AsLOV2 (from Aveva sativa), have also appeared, all with desirably sufficient 1O2 quantum yields [Different variants of KillerRed and miniSOG have also appeared, either to monomerize the KillerRed dimer (SuperNova and GreenSuperNova) ,45, to br SOPP3) ,50,51,52 [Q103V) . A red fum yield . 1O2-genm yields ,57,58,59In view of the above developments, it has become pertinent to examine whether the newly emerged GEPP could also be used for photodynamic CCK1R activation. Further, numerous works have reported that G protein-coupled receptors (GPCR) function not only from PM but, also, from intracellular membranes ,61. The C71G, and DsFbFP in AR4-2J cells after light irradiation all photodynamically activated endogenous CCK1R in the rat pancreatic acinar tumor cell AR4-2J, triggering persistent calcium oscillations, with DsFbFP being the least effective. Interestingly, light irradiation of AR4-2J cells with miniSOG expressed at the plasma membrane (PM), mitochondria (MT), or lysosomes (LS) was found to trigger similarly persistent calcium oscillations, which were inhibited by CCK1R antagonist devazepide 2 nM with a graded sensitivity of PM > MT > LS. Therefore, GEPP expressed either at PM or intracellularly are both effective to photodynamically activate CCK1R, suggesting that photodynamic action may be particularly suited for the study of intracellular GPCR without the need for extracellularly added agonists to overcome multiple diffusion barriers. In the present work, it was found that plasma membrane (PM)-expressed KillerRed, miniSOG, miniSOG2, SOPP, Mr4511PM vector was bought from Evrogen . Ampicillin and kanamycin were from CWBio . Endotoxin-free plasmid extraction kit and DH5à competent cells were from TianGen Biochemicals . MitoTracker™ Red FM was from Invitrogen . LysoTracker Red was from Beyotime . Sulfated cholecystokinin octapeptide (CCK) and CCK1R antagonist devazepide were from Tocris Cookson . Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium was bought from Invitrogen . Fura-2 AM was from AAT Bioquest . JetPRIME transfection reagent was from PolyPlus-transfection SA . Fetal bovine serum (FBS) was from Thermo Scientific . pKillerRed2 incubator under humidified atmosphere (5% CO2/95% air) at 37 °C, as reported previously [AR4-2J was bought from The American Type Culture Collection  and cultured in DMEM/F12 supplemented with 20% fetal bovine serum in a COeviously ,62,63,64E. coli medium LB/kana and LB/amp were sterilized and culture plates made. Liquid E. coli medium LB/kana and LB/amp had the same composition but without agar. Solid PMpKillerRed was bought from Evrogen , proliferated in and harvested from competent E. coli. A mammalian codon-optimized miniSOG gene (GenBank accession number JX999997) was synthesized de novo from nucleotides at Genscript  with the following full sequence: ATGGAAAAGAGCTTTGTGATTACCGATCCGCGCCTGCCAGACAACCCGATCATTTTCGCGAGCGATGGCTTTCTGGAGTTAACCGAATATTCTCGTGAGGAAATTCTGGGTCGCAATGGCCGTTTCTTGCAGGGTCCGGAAACGGATCAAGCCACCGTGCAGAAAATCCGCGATGCGATTCGTGACCAACGCGAAATCACCGTTCAGCTGATTAACTATACGAAAAGCGGCAAGAAATTTTGGAACTTACTGCATCTGCAACCGATGCGCGATCAGAAAGGCGAATTGCAATATTTCATTGGTGTGCAGCTGGATGGCTAG. This synthesized full miniSOG gene sequence was inserted into plasmid PMpKillerRed  to replace the KillerRed sequence. Competent E. coli were infected with the recombinant plasmid, cultured on solid LB/kana. Bacteria colonies were picked and further cultured in liquid LB/kana with shaking overnight. Proliferated plasmid was extracted with sequence verification. The plasmid so obtained was named PMpminiSOG due to the presence of the PM-localization sequence in the original Evrogen plasmid. After transfection with plasmid PMpKillerRed or PMpminiSOG, positive expressing AR4-2J cells were named KillerRedPM- or miniSOGPM-AR4-2J cells, as reported before [Plasmid d before ,32. PMpminiSOG2, the miniSOG2 [miniSOG sequence in plasmid PMpminiSOG was replaced with the synthesized miniSOG2 sequence to obtain plasmid PMpminiSOG2 . The miniSOG2 sequence was: ATGGAGAAGAGCTTCGTGATCACCGACCCCCGCCTGCCTGACAACCCAATCATCTTCGCCAGCGACTCCTTCCTGGAGCTGACCGAGTACTCCAGGGAGGAGATCCTGGGAAGGAACCCACGGTTCCTGAGAGGACCTGAGACCGACCAGGCAACCGTGCAGAAGATCCACGACGCCATCCGCGACCAGAGGGAGATCACCGTGCAGCTGATCAACTACACCAAGAGCGGCAAGAAGTTCTGGAACCTGTTCCGGCTGCAGCCAATCAGAGACCAGAAGGGCGAGCTGCAGTACTTCATCGGCGTGCAGCTGGACGGCTAA. AR4-2J cells transfected with plasmid PMpminiSOG2 were named miniSOG2PM-AR4-2J cells. For the construction of plasmid miniSOG2  gene wasPMpSOPP, the SOPP amino acid sequence [SOPP gene was synthesized de novo after rat codon optimization. The miniSOG sequence in plasmid PMpminiSOG was replaced to obtain plasmid PMpSOPP , where the SOPP gene sequence was: ATGGAGAAGAGCTTCGTGATCACCGACCCCAGGCTGCCTGACAACCCAATCATCTTCGCCAGCGACGGCTTCCTGGAGCTGACCGAGTACTCCAGGGAGGAGATCCTGGGAAGGAACGGCCGGTTCCTGCAGGGACCCGAGACCGACCAGGCCACCGTGCAGAAGATCAGAGACGCCATCAGAGACCAGCGCGAGATCACCGTGCAGCTGATCAACTACACCAAGTCCGGCAAGAAGTTCTGGAACCTGCTGCACCTGCAGCCCATGCGGGACCAGAAGGGCGAGCTGCAGTACTTCATCGGCGTGCTGCTGGACGGCTAA. AR4-2J cells transfected with plasmid PMpSOPP as verified were named SOPPPM-AR4-2J cells.For the construction of plasmid sequence  was usedC71GPMpMr5411, the Mr5411C71G protein sequence from [C71GMr5411 gene was synthesized de novo with rat codon optimization, which was used to replace the miniSOG sequence in PMpminiSOG . The C71GMr5411 sequence was ATGGAGACCGGAGGAACCGCCACCAGCCACGTGCCAGACGAGCTGAAGGCAGAGTCCCACAGAGGCGACCCTTTCGCCGCAGCCGTGAGGGCAACCAGGATGCCCATGATCATCACCGACCCTGCCCAGCACGACAACCCAATCGTGTTCGTGAACGACGCCTTCCTGAAGCTGACCGGCTACACCAGGATGGAGGTGGTGGGAAGAAACGGCCGCTTCCTGCAGGGACCAGACACCGAGGCAGCAGCAGTGGACAGACTGAGGGCAGCCATCAGGCGGGAGGAGGACATCAGAGTGGACCTGCTGAACTACCGCAAGGACGGCAGCACCTTCCAGAACGCCCTGTACGTGGGACCCGTGAGGGACGAGGCAGGACGGGTGGTGTACTTCTTCGCCAGCCAGCTGGACGTGTCCGAGCACTACGCCCTGACCGCAGAGATCGAGAGGCTGAAGGCCGCCCTGGCCGAGGCCGAGGCCAAGCTGGCCGCCCGGTAG. AR4-2J cells transfected with plasmid C71GPMpMr5411 were named C71GPMMr5411-AR4-2J cells.For the construction of plasmid nce from  was usedDsFbFP gene [miniSOG from plasmid PMpminiSOG . The DsFbFP gene sequence was ATGAGGCGGCACTACCGCGACCTGATCAGGAACACCCCCATGCCTGACACCCCACAGGACATCGCAGACCTGCGCGCCCTGCTGGACGAGGACGAGGCCGAGATGAGCGTGGTGTTCAGCGACCCATCCCAGCCCGACAACCCTATGATCTACGTGTCCGACGCCTTCCTGGTGCAGACCGGATACACCCTGGAGGAGGTGCTGGGAAGGAACGCAAGATTCCTGCAGGGACCAGACACCAACCCACACGCAGTGGAGGCAATCAGGCAGGGCCTGAAGGCAGAGACCAGATTCACCATCGACATCCTGAACTACAGGAAGGACGGCAGCGCCTTCGTGAACAGACTGCGCATCAGGCCTATCTACGACCCAGAGGGCAACCTGATGTTCTTCGCCGGCGCCCAGAACCCCGTGCTGGAGTAG. Positive AR4-2J cells after transfection with plasmid PMpDsFbFP were named DsFbFPPM-AR4-2J cells.The bFP gene  was syntMTpminiSOG was prepared by replacing the PM-localizing sequence ATGCTGTGCTGTATGAGAAGAACCAAACAGGTTGAAAAGAATGATGAGGACCAAAAGATC in PMpminiSOG with the mitochondrial (MT)-targeting sequence (MTS: ATGTCCGTCCTGACGCCGCTGCTGCTGCGGGGCTTGACAGGCTCGGCCCGGCGGCTCCCAGTGCCGCGCGCCAAGATCCATTCGTTGGGGGATCCACCGGTCGCCACC) . LSpminiSOG was prepared by replacing the PM-localizing ATGCTGTGCTGTATGAGAAGAACCAAACAGGTTGAAAAGAATGATGAGGACCAAAAGATC sequence in PMpminiSOG with the lysosomal (LS) sequence (LTS: ATGAAGGGACAGGGAAGCATGGACGAGGGAACCGCCGACGAGAGGGCCCCCCTGATCCGGACC). Competent E. coli were infected with the plasmid, further cultured on solid LB/kana. Bacteria colonies were picked and cultured in liquid LB/kana with shaking overnight. Propagated plasmids were extracted for sequence verification. The plasmid constructs were designated MTpminiSOG and LSpminiSOG; transformed AR4-2J cells were named miniSOGMT-AR4-2J and miniSOGLS-AR4-2J cells, accordingly.The plasmid ex 543 nm for KillerRedPM, and λex 488 nm for miniSOGPM, miniSOG2PM, Mr4511C71GPM, and DsFbFPPM. Mitochondrial or lysosomal expressions of miniSOG (λex 488 nm) were verified by colocalization with MitoTracker Red or LysoTracker Red (λex 543 nm) in confocal imaging (Zeiss LSM510 META), objective × 60 oil. AR4-2J cells were cultured in 6-well plates with one round glass cover-slip in each well, and to be transfected, cells were allowed to grow to 50–70% confluence. Plasmid (2 μg/well) and JetPRIME transfection reagent (4 μL/well) in JetPRIME buffer (200 μL) were added; then, AR4-2J cells were cultured for a further 24 h. Positive cellular GEPP expression was verified by confocal imaging of GEPP fluorescence: λPM-AR4-2J cells. AR4-2J cells cultured in a Petri dish (35 mm) were transfected; twenty-four hours later, cells were washed in PBS before the extraction of RNA. RNA concentration was determined with a Nanodrop2000 nanospectrometer . mRNA was reverse-transcribed with a GoScript Reverse Transcription Kit A5001  to obtain cDNA. To a PCR tube was added Oligo (dT) 1 μL, RNA 1 μg, 70 °C denaturation for 5 min, cooled on ice for 5 min, before the addition of the reaction buffer (×5) 5 μL, RNAase inhibitor 1 μL, M-MLV reverse transcriptase 1 μL, dNTP (10 mM) 1.25 μL, topped up with diethyl pyrocarbonate (DEPC)-treated water to 25 μL. Reverse transcription conditions: 40 °C, 60 min and 70 °C, 15 min to obtain cDNA. PCR reaction: 2-μL cDNA template, 15-μL 2×Taq Master Mix , primers 1 μL each, topped up to 30 μL. Initial de-naturation 95 °C, 5 min; PCR cycles: 94 °C, 30 s, 60 °C, 30 s, 72 °C, 1.5 min, 30 cycles, and final prolongation 72 °C, 5 min. The RT-PCR product was run on 1% agarose gel with 0.01% GoodView  added, 120 V, 40 min before imaging. PCR primers for DsFbFP were: forward 5′-GGCACTACCGCGACCTGATC-3′ and reverse 5′-CTACTCCAGCACGGGGTTCT-3′. Primers for internal reference GAPDH were: forward 5′-GTGGAGTCTACTGGCGTCTT-3′ and reverse 5′-CCAGGATGCCCTTTAGTG-3′.RT-PCR (reverse transcription-polymerase chain reaction): HiPure Total RNA Plus Mini Kit  was used as instructed in the manufacturer’s manual for RNA extraction from AR4-2J and DsFbFPPM-AR4-2J cells were irradiated with white light  from a halogen cold light source  equipped with a condenser (HLL201). AR4-2J cells expressing PM-localizing miniSOG, miniSOG2, SOPP/miniSOGQ103L, Mr4511C71G, DsFbFP, MT- or LS-localizing miniSOG were irradiated with blue LED  . Power density was measured at the level of attached cells in the Sykes-Moore perfusion chamber with a power meter . Light-responding transfected AR4-2J cells were identified as GEPP-positive cells. KillerRed340/F380 and plotted against time with SigmaPlot , as reported before [N  identical experiments.Parental control or transfected AR4-2J cells grown on glass cover-slips in 6-well plates were loaded with Fura-2 AM  for 1 h after assembly in the Sykes-Moore perfusion chamber. Cytosolic calcium was measured in an inverted fluorescent microscope (Nikon TE-2000U)  coupled to a Photon Technology International  calcium measurement system with alternating excitations at 340 nm/380 nm (DeltaRam X); emitted Fura-2 fluorescence  was detected with a charge-coupled device (CCD) camera . Calcium concentration was expressed as Fura-2 fluorescence ratios Fd before ,63,64,66N (as indicated) independent experiments were presented in bar graphs as mean ± SEM, unless specifically stated otherwise. Student’s t-test was used for statistical analysis against controls, and p < 0.05 was taken as significant and indicated with an asterisk (*).All calcium tracings and other graphs were plotted with SigmaPlot. For calculation and comparison of the strength of induced calcium responses, the calcium peak area above the baseline was integrated . Statistical data from PMpKillerRed, PMpminiSOG, PMpminiSOG2, PMpSOPP, C71GPMpMr4511, and pDsFbFP , DsFbFP demonstrated markedly dimmer fluorescence, suggesting poorer protein expression, as shown in the confocal images  in parental AR4-2J cells . In parallel experiments, white light irradiation  from the halogen light source was found to induce sustained calcium oscillations in KillerRedPM-AR4-2J cells  elicited persistent calcium oscillations in miniSOGPM-  like previously reported ,32, we niniSOGLS a, with aoTracker b,c. −2, 1.5 min) was found to have no effect on the baseline calcium level in parental AR4-2J cells, but in these cells, CCK 20 pM induced marked calcium responses  induced long-lasting calcium oscillations in miniSOGMT-AR4-2J cells  similarly induced long-lasting calcium oscillations in miniSOGLS-AR4-2J cells  identical experiments was calculated and plotted as bar graphs (i). Note the sharp difference in the calcium response after LED light irradiation in parental AR4-2J cells, and dark or light responses in MT or LS miniSOG-transfected AR4-2J cells (i). Quantitative analysis of calcium responses in original tracings, as represented in PM-AR4-2J cells  were inhibited nearly completely by the CCK1R antagonist devazepide 2 nM  but no effect on miniSOGLS photodynamic action  . For the representative experiment shown in MT photodynamic action was inhibited without significance by devazepide 2 nM (from 100% to 65%) but significantly by devazepide 10 nM (from 100% to 29%)  d. miniSO to 29%) d. C71G, and DsFbFP after light irradiation all photodynamically activated CCK1R to induce persistent cytosolic calcium oscillations in AR4-2J cells, but the photodynamic effect of DsFbFP was much reduced in comparison, likely due to poor protein expression. Permanent photodynamic CCK1R activation was achieved in AR4-2J cells by miniSOG expression not only at the plasma membrane (PM) but, also, in mitochondria (MT) and lysosomes (LS). Calcium oscillations induced by miniSOG photodynamic action at intracellular sites showed reduced sensitivity to inhibition by CCK1R antagonist devazepide 2 nM with the order of PM > MT > LS. In the present work, it was found that PM-expressed KillerRed, miniSOG, miniSOG2, SOPP, Mr45111O2 generated in type II photodynamic action with SALPC, KillerRed, or miniSOG as the photosensitizer [−2, 4 min and miniSOG with blue LED 450 nm, 85 mW‧cm−2, 1.5 min) triggered long-lasting calcium oscillations in both KillerRedPM-AR4-2J and miniSOGPM-AR4-2J cells (Q103L), Mr4511C71G, and DsFbFP expressed at the plasma membrane  [bl50% miniSOG: 2.85 min and DsFbFP: 0.35 min) [PM-AR4-2J cells after blue LED irradiation (Dinoroseobacter shibae) has been known to be expressed in CHO-K1 cells as a dimer, which might also affect its fluorescence, unlike the monomeric miniSOG, miniSOG2, or SOPP, all sourced from Arabidopsis thaliana [Although the DsFbFP protein level was low, as shown by the dim DsFbFP fluorescence, DsFbFP mRNA were expressed at sufficient levels in AR4-2J cells, as verified by RT-PCR experiments g. Althou = 0.35)  is the h.35 min) . This waadiation g. DsFbFPthaliana ,70. The thaliana  might fa1O2 after light irradiation in a type II photodynamic action [1O2 of 0.03 (as reviewed in [2−.) [1O2 [1O2 than O2−., with an ϕ1O2 of 0.008, whilst monomeric KillerRed SuperNova has an ϕ1O2 of 0.02 [SOPP or miniSOGQ103L) has a ϕ1O2 of 0.25 [C71G sourced from Methylobacterium radiotolerans has a ϕ1O2 of 0.19 [1O2 among the lot at 0.33 [1O2 from 0.008 to 0.33 . Killerin [2−.) , but mor−.) [1O2 ,36,43. P of 0.02 . miniSOG of 0.02 . The sin of 0.25 . Mr4511C of 0.19 . DsFbFP  at 0.33 . Althoug0.33 see , no signefficacy . This in−2, 1 min) induced phototoxicity in a small percentage of cells two hours after light irradiation of miniSOG-HEK293, miniSOG2-HEK293, and SOPP-HEK293 cells, but no marked difference was found in HEK293 cells expressing SOPP (9%) with a ϕ1O2 of 0.25 or miniSOG (11%) with a ϕ1O2 of 0.03 [1O2 of only 0.03 to permanently activated photodynamically CCK1R in miniSOGPM-AR4-2J cells in the present work might be related to the progressive photochemical transformation of the fluorophore flavin mononucleotide (FMN) to lumichrome and the photo-oxidization of internal residues in miniSOG to significantly increase its ϕ1O2 up to 10-fold [1O2 is not determined; see Avena sativa phototropin 1, light irradiation was found to induce progressive photochemical dissociation or the release of FMN from the AsLOV2 protein moiety, leading to significantly increased ϕ1O2 [It has been found by others that blue LED irradiation  of the visible spectrum (full-spectrum white light was used in the present work), whereas all others by blue light (450 nm)   (for the(450 nm) . Althoug(450 nm)  might waC71G, and DsFbFP. It would be ideal if KillerRed could be subjected to further annotations, to shift its maximal excitation peak toward even longer wavelengths (a red shift), possibly by genetic code expansion [C71G, DsFbFP -induced calcium oscillations were inhibited by CCK1R antagonist devazepide 2 nM significantly in miniSOGPM-CHO-K1 cells, slightly in miniSOGMT-AR4-2J cells, but not at all in miniSOGLS-AR4-2J cells  or lysosomal (LS) miniSOG were found to photodynamically trigger persistent calcium oscillations similarly . LED irr2J cells , althoug2J cells . The cal2J cells . We beli present ,76,77. S present  and are  present ; therefonometers  could eaLS photodynamic CCK1R activation and the little inhibition afforded by CCK1R antagonist devazepide in miniSOGLS-AR4-2J cells. The insensitivity of lysosomal CCK1R to the antagonist devazepide 2 nM might also be due to limited accessibility of lysosomal CCK1R to extracellularly added devazepide 2 nM. Such a reduced sensitivity of intracellular GPCR to ligands was noted before for the nuclear membrane GPCR in cardiomyocytes, for example [The lysosomal accumulation of endocytosized CCK1R and partial CCK1R degradation ,79,80,81 example . 1O2 generation, due to its limited lifetime of 1 µs [1O2 in the cellular milieu has been suggested to be in the tens of nanometers or more (20–150 nm) [1O2 could diffuse from the plasma membrane (PM), mitochondrial (MT), or lysosomal (LS) membranes as the origin to within a circle with a radius of 20–150 nm. Although the emphasis in the present work was the subcellular localization of miniSOG expression and therefore of subcellular  of 1 µs ,84,85 th–150 nm) ,86,87,881O2 could well help to further investigate GPCR functions at these intracellular sites. Photodynamic GPCR activation/modulation might offer distinct advantages over conventional receptor pharmacology in that no ligand is needed for photodynamic activation after GEPP  expression at defined intracellular sites. Only light irradiation is required to permanently activate the intracellular GPCR. The limited diffusion distance of photodynamically generated 1O2 (20–150 nm) may ensure spatial precision and specificity. It may be noted that there is abundant evidence for GPCR expression, localization, and function at nuclear , mitocho1O2 activation of CCK1R. In the present work, all GEPP examined were found to elicit persistent calcium oscillations  photodynamically, either from the PM, MT, or LS. We have previously found that the SALPC photodynamic activation of CCK1R in rat pancreatic acini involved the near quantitative transformation of the CCK1R protein dimer to the monomer . In addiC71G, and DsFbFP) reported in the literature were found to photodynamically activate the endogenous CCK1R in AR4-2J cells after plasma membrane expression. The miniSOG expression at intracellular sites was also found to induce persistent calcium oscillations or CCK1R activation (In conclusion, representative GEPP (KillerRed, miniSOG, miniSOG2, SOPP, Mr4511tivation . The pre


Caenorhabditis elegans, the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in C. elegans female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I.Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore–microtubule attachments in yeast and mouse meiosis. In  Formation of a diploid embryo requires that sperm and egg contribute exactly one copy of each chromosome. The cell division in charge of reducing ploidy of the genome is meiosis, which involves two chromosome segregation steps after a single round of DNA replication . Female Protein Phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase composed of a catalytic subunit C (PPP2C), a scaffolding subunit A (PPP2R1), and a regulatory subunit B (PPP2R2–PPP2R5) . While tPP2A:B56 can be targeted to distinct sites by different proteins, and two mechanisms have been characterised at the structural level. While an N-terminal coiled coil domain in Shugoshin/MEI-S332 binds PP2A:B56 , other sCaenorhabditis elegans, the meiotic role of PP2A remains unexplored. The core components of the PP2A holoenzyme in C. elegans are LET-92  and PAA-1 (scaffolding A subunit), and the regulatory B subunits are B55SUR-6  and paa-1(RNAi), oocytes microtubules do not organise into a bipolar structure (let-92(RNAi) oocytes, displaying a cluster of small foci  activity (i.e. anaphase progression). Cytoplasmic securinIFY-1 degradation proceeded at similar rates in wild-type and LET-92-depleted oocytes  in vivo, we attempted to tag endogenous LET-92 with GFP but were unsuccessful, presumably due to disruption of its native structure, in agreement with a recent report . We geneeiosis I . The fuseiosis I , in agreeiosis I . During eiosis I .C. elegans B56 orthologues, PPTR-1 and PPTR-2, and the single B55 orthologue, SUR-6  during metaphase of meiosis I  with RNAi-mediated depletion of B56αPPTR-1 (‘pptr-1(RNAi)’), which we will refer to hereafter as ‘PPTR-1/2 depletion’. While there was no significant change in GFP::PAA-1 localisation upon depletion of B56αPPTR-1 or deletion of B56γPPTR-2, no PAA-1 signal is detected associated with chromosomes upon double PPTR-1/2 depletion  does not inhibit B56 localisation  involved in targeting PP2A:B56 to meiotic chromosomes. Two of the most studied proteins involved in B56 targeting are Shugoshin and BubR1. The BubR1 orthologue, Mad3 spindle  and Mad3lisation . We thery CRISPR  and obse1 levels  and a sl2 levels . ParticuC. elegans B56 subunits display a dynamic localisation pattern similar to that of the kinase BUB-1 throughout meiosis I   . Sequenc 282–287 . When co 282–287 . In addi elegans . The put elegans , with seL282A,V285A was indistinguishable from that of wild-type BUB-1 during metaphase I , but segregation was achieved in 100% of the cases with no PBE defects were detected. Interestingly, the newly identified LxxIxE motif specifically recruits PP2A:B56 to metaphase chromosomes and anaphase central spindle, but not anaphase chromosomes.Using a fluorescence polarisation-based assay, we confirmed that the LxxIxE motif of BUB-1 binds to purified recombinant PPTR-2, and this binding is abolished by the L282A,V285A mutations . While laphase I , localisA mutant . B56γPPTxE motif . In cont spindle  without  spindle . Some GFteractor , is not teractor . Mutatioteractor , indicatPPTR-2 with higher affinity than non-phosphorylated LxxIxE peptide  had a similar effect to that of the BUB-1L282A,V285A mutant: it significantly reduced B56αPPTR-1 localisation  destabilises the kinase domain and prevents its interaction with Mad1MDF-1  the two pools of B56γPPTR-2.These results show that the BUB-1 kinase domain is important for recruitment of B56γC. elegans. PP2A is essential for meiosis I: depletion of the catalytic or scaffold subunits leads to severe spindle assembly defects, lack of chromosome segregation, and failure to achieve PBE. These effects are likely brought about by a combination of different PP2A subcomplexes with varying regulatory B subunits. PP2A:B56 regulates chromosome dynamics prior to segregation since depletion of the two B56 orthologues, PPTR-1 and PPTR-2, leads to alignment defects. We have uncovered a new phospho-regulated B56 LxxIxE motif in C. elegans BUB-1 that recruits PP2A:B56 and is important for chromosome alignment during meiosis I.In the present article, we have uncovered new roles for PP2A during oocyte meiosis in Depletion of the sole catalytic or scaffold PP2A subunits leads to massive meiotic failure and embryonic lethality. The earliest effect we could see in our experimental set up is a failure to assemble a bipolar spindle. This will of course have a direct impact on any process that should occur after spindle assembly, including chromosome alignment and segregation, followed by PBE. However, BUB-1 depletion leads to severe spindle assembly and alignment defects, yet in the majority of cases, chromosomes segregate (with visible errors – lagging chromosomes) and polar bodies do extrude. Therefore, lack of a proper bipolar metaphase spindle is not sufficient to result in lack of segregation or PBE. PP2A complexes harbouring the B56 subunits PPTR-1 and PPTR-2 are required to target the phosphatase to chromosomes to regulate chromosome alignment in metaphase I. Furthermore, this chromosomal B56 pool is recruited by BUB-1 through its LxxIxE SLiM motif. Mutation of this motif that prevents binding to B56 subunits leads to alignment defects. B56 subunits however are not involved in spindle pole targeting of PP2A, suggesting that this spindle pole pool is the one relevant for spindle assembly. Our attempts to address a possible role for other B subunits were focused on RSA-1 and SUR-6 given their reported centrosomal roles during mitosis . HoweverC. elegans oocytes is highly dynamic, and differences have been observed between metaphase and anaphase. For example, while kinetochore localisation of the CLASP orthologue CLS-2 during metaphase depends on BUB-1, a pool of CLS-2 is able to localise in the anaphase central spindle in a BUB-1-independent manner  resemble those reported in the absence of kinetochore proteins . InteresC. elegans, the Aurora B orthologue, AIR-2, concentrates in the interface between homologous chromosomes  closely resembling the consensus for PP2A:B56 reported in During meiosis in bivalent . Consistbivalent . During tochores  and it iC. elegans BUB-1 .Strains used in this study were maintained at 20 degrees unless indicated otherwise. For a complete list of strains, please refer to For RNAi experiments, we cloned the different sequences in the L4440 RNAi feeding vector .600 of 0.6–0.8. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and cultures were incubated overnight at 20°C. Bacteria were then seeded onto NGM plates made with agarose and allowed to dry. L4 worms were then plated on RNAi plates and grown to adulthood at 20°C for 24 hr in the case of let-92(RNAi), bub-1(RNAi), and paa-1(RNAi) and 48 hr in all other cases.All sequences were inserted into L4440 using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) and transformed into DH5a bacteria. The purified plasmids were then transformed into HT115(DE3) bacteria . RNAi clFor the generation of in situ-tagged GFP::SUR-6, we used the self-excising cassette method . In brieK718R,D847N, BUB-1L282A,V285A, BUB-1S283A, and BUB-1L282A,V285A, K718R,D847N were generated by Sunybiotech.The strains AID::GFP::GSP-2, GFP::PAA-1, BUB-1.CTCGACAATATCATCTCCAGATTATTGGAAGATGcctaaagatccagccaaacctccggccaaggcacaagttgtgggatggccaccggtgagatcataccggaagaacgtgatggtttcctgccaaaaatcaagcggtggcccggaggcggcggcgttcgtgaagAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagCCAACACTTGTCACTACTTTCTgTTATGGTGTTCAATGCTTcTCgAGATACCCAGATCATATGAAACgGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAAGACGTAGAAAAGCTTAAT..GCTACTGACGACGCGATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagCCAACACTTGTCACTACTTTCTgTTATGGTGTTCAATGCTTcTCgAGATACCCAGATCATATGAAACgGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAAGGAGGTGGATCCGGTGGTGGATCCTCGGTTGTCGAAGAA.The wild-type sequence:AAGTACGAGGTGCCATCATGTTCGTGGGAAGTGTACATTTGCGACCAAATGCGGAATCGCCTGAAAGATCGAGGTTTGGAGCTGATGGCCAAATGTTGCATTATGGAAGTGATGGATGCTTATGTTTATTCAACTGCTTCGCTTCTTGTTAATCAGTACCACGAATATGGAACGCTGCTTGAATATGCGAATAACATGAAGGATCCGAATTGGCACATAACCTGCTTCTTGATTACCCAAATGGCCCGAGTTGTGAAGGAAGTCCATGCCTCTAAAATTATTCATGGAGATATCAAACCGGATAATTTTATGATCACCAGAAAgtatgggaaaacatttgttaattttagacgttatcttttttcagGATCGATGATAAATGGGGCAAAGATGCTCTGATGAGTAACGACAGCTTTGTCATCAAGATTATCGACTGGGGACGTGCCATTGACATGATGCCACTGAAGAACCAGCGTGTAACCGATGATCAAAGGACAGTAGCTGTGwas mutated to:CGCTACGAGGTGCCATCATGTTCGTGGGAAGTGTACATTTGCGACCAAATGCGGAATCGCCTGAAAGATCGAGGTTTGGAGCTGATGGCCAAATGTTGCATTATGGAAGTGATGGATGCTTATGTTTATTCAACTGCTTCGCTTCTTGTTAATCAGTACCACGAATATGGAACGCTGCTTGAATATGCGAATAACATGAAGGATCCGAATTGGCACATAACCTGCTTCTTGATTACCCAAATGGCCCGAGTTGTGAAGGAAGTCCATGCCTCTAAAATTATTCATGGAGATATCAAACCGGATAATTTTATGATCACCAGAAAgtatgggaaaacatttgttaattttagacgttatcttttttcagGATCGATGATAAATGGGGCAAAGATGCTCTGATGAGTAACGACAGCTTTGTCATCAAGATTATCAATTGGGGACGTGCGATTGACATGATGCCACTGAAGAACCAGCGTGTAACCGATGATCAAAGGACAGTAGCTGTGThe introduced changes are shown in red, and synonymous mutations are shown in cyan.The wild-type sequence:CTTTCACCAGTCAGTGAGAAAACGGTTGATGATGAGGAGGAAAAGAACGCCAATCTAAATCCTAGAAGACGTCATwas mutated to:GCATCACCAGCTAGCGAGAAAACGGTTGATGATGAGGAGGAAAAGAACGCCAATCTAAATCCTAGAAGACGTCATThe wild-type sequence:CATCTTTCACCAGTCAGTGAGAAAACGGTT GATGATGAGTTCAACGCCAATCTAAATCCTAGAAGACGTwas mutated to:CACCTTGCGCCAGTCAGTGAGAAAACGGTTGATGATGAGTTCAACGCCAATCTAAATCCTAGAAGACGTThe introduced changes are shown in red, and synonymous mutations are shown in cyan.See The wild-type sequence:CTTTCACCAGTCAGTGAGAAAACGGTTGATGATGAGGAGGAAAAGCGAAGCCGGATTTATTCGCCGCTGGTTGCAACGAAGGATGCTCACAGACCTGCACTTCGGAGCAAAATTGAGAATCCTCCAGCGACAGTGACACTTTCGTCGGATACAAAGTCTGCTTCGGAGAAAGATGTTAGTGATTCCGATGATGCAGATGATGATGAAAGACTCAAGATTATGACTGCCGGCAGAAAAGATGGTAACCCTCCAGACCGTTCCACAAGCATATCTTCCAACTATTCAACTGCTTCTGCAAGAACATCAAAGAGTGGAGCTGGATTGGATTTGATGGCGGAAAATAAGTGTTTGGAGGCACATGCTATGTTTTCCGACACTGTACATCTTGCTAGCGAAAAGACAATGGTCCTTGGCGATGATTCTGTCTTCGTTCCAGAAAGATCTTTAGCTACTACGCAGATAGTTACTGACTTTTCCGTGCTCTGTGATCCTGATCCGACAATGACCATTACACAGGAGCGTCCGAAAAAAGTGTCGAATGGGTTGAATGTTGTTTACGATGAGGCAGCCGAACCGGAAGAATCTCAGAAAGTTGAGGAATCTGAAGTACAACCCGAAATTGTCCTAGTTTCTCCAGTGACGCAAACCTCACCAGCTACAATGTTTAATGATAGTGGGTTTATCGAAAAATATAACAACTTATGTTTTAATTTTTAGTTTATGACGATGAAATCGAGTTTGGCTTTTTCAAACCGTCTCGTGGTAATTTCGTCACATCGACCCCCGCACAAGGAGTTCATTTGGTCAACATTGATGAATATTTCGGAAATAAAGAGGAGGAAAGCACTCACGAACAGGAAGCTCCAGTATTTGTTGCTCCAACCAGCAGTACTTTCAGTAAATTAGTAAGTGCCAGACAAATTTTCGACATACTATTCAAACTTTTTCAGACACGTCGAAAGTCACTAGCAGCAAATCAAGCCGTTCAGCCCTCAGTCACAGAGTCATCAAAGCCTGAACGATCAGATCCTAAAGATTCATCTATCGATTGTTTGACAGCTAATCTAGGAAGACGTCTTTCAATTGGTGCTGATGAAATTCCAAATCTCACTGAAAACAACGAATCTGAAATCACTGGTTGCAAGATTCGTCGGCGCAGTGAAATTATCAAGCAAGGAGACATCAATCCATGGGACGAAACTCTTCGAAAAAAATTGATGTGTCTTGTGCGTCCTCCCCAGAATATGCACGAGTTCCAAGAACGAGCACCGAAGATTCAAGCTCTGAGAGACTGCGAGGTTAGCGGAGAAAAGCTCCACATTCAAACTCTTATTGGTCAAGGTGGATACGCTAAAGTATACCGGGCTGTAACCGATGATCAAAGGACAGTAGCTGTGAAGTACGAGGTGCCATCATGTTCGTGGGAAGTGTACATTTGCGACCAAATGCGGAATCGCCTGAAAGATCGAGGTTTGGAGCTGATGGCCAAATGTTGCATTATGGAAGTGATGGATGCTTATGTTTATTCAACTGCTTCGCTTCTTGTTAATCAGTACCACGAATATGGAACGCTGCTTGAATATGCGAATAACATGAAGGATCCGAATTGGCACATAACCTGCTTCTTGATTACCCAAATGGCCCGAGTTGTGAAGGAAGTCCATGCCTCTAAAATTATTCATGGAGATATCAAACCGGATAATTTTATGATCACCAGAAAGTATGGGAAAACATTTGTTAATTTTAGACGTTATCTTTTTTCAGGATCGATGATAAATGGGGCAAAGATGCTCTGATGAGTAACGACAGCTTTGTCATCAAGATTATCGACTGGGGACGTGCCATTGACATGATGCCACTGAAGAACCAGCGTAACGCCAATCTAAATCCTAGAAGACGTCATwas mutated to:GCATCACCAGCTAGCGAGAAAACGGTTGATGATGAGGAGGAAAAGCGAAGCCGGATTTATTCGCCGCTGGTTGCAACGAAGGATGCTCACAGACCTGCACTTCGGAGCAAAATTGAGAATCCTCCAGCGACAGTGACACTTTCGTCGGATACAAAGTCTGCTTCGGAGAAAGATGTTAGTGATTCCGATGATGCAGATGATGATGAAAGACTCAAGATTATGACTGCCGGCAGAAAAGATGGTAACCCTCCAGACCGTTCCACAAGCATATCTTCCAACTATTCAACTGCTTCTGCAAGAACATCAAAGAGTGGAGCTGGATTGGATTTGATGGCGGAAAATAAGTGTTTGGAGGCACATGCTATGTTTTCCGACACTGTACATCTTGCTAGCGAAAAGACAATGGTCCTTGGCGATGATTCTGTCTTCGTTCCAGAAAGATCTTTAGCTACTACGCAGATAGTTACTGACTTTTCCGTGCTCTGTGATCCTGATCCGACAATGACCATTACACAGGAGCGTCCGAAAAAAGTGTCGAATGGGTTGAATGTTGTTTACGATGAGGCAGCCGAACCGGAAGAATCTCAGAAAGTTGAGGAATCTGAAGTACAACCCGAAATTGTCCTAGTTTCTCCAGTGACGCAAACCTCACCAGCTACAATGTTTAATGATAGTGGGTTTATCGAAAAATATAACAACTTATGTTTTAATTTTTAGTTTATGACGATGAAATCGAGTTTGGCTTTTTCAAACCGTCTCGTGGTAATTTCGTCACATCGACCCCCGCACAAGGAGTTCATTTGGTCAACATTGATGAATATTTCGGAAATAAAGAGGAGGAAAGCACTCACGAACAGGAAGCTCCAGTATTTGTTGCTCCAACCAGCAGTACTTTCAGTAAATTAGTAAGTGCCAGACAAATTTTCGACATACTATTCAAACTTTTTCAGACACGTCGAAAGTCACTAGCAGCAAATCAAGCCGTTCAGCCCTCAGTCACAGAGTCATCAAAGCCTGAACGATCAGATCCTAAAGATTCATCTATCGATTGTTTGACAGCTAATCTAGGAAGACGTCTTTCAATTGGTGCTGATGAAATTCCAAATCTCACTGAAAACAACGAATCTGAAATCACTGGTTGCAAGATTCGTCGGCGCAGTGAAATTATCAAGCAAGGAGACATCAATCCATGGGACGAAACTCTTCGAAAAAAATTGATGTGTCTTGTGCGTCCTCCCCAGAATATGCACGAGTTCCAAGAACGAGCACCGAAGATTCAAGCTCTGAGAGACTGCGAGGTTAGCGGAGAAAAGCTCCACATTCAAACTCTTATTGGTCAAGGTGGATACGCTAAAGTATACCGGGCTGTAACCGATGATCAAAGAACAGTAGCTGTGCGCTACGAGGTGCCATCATGTTCGTGGGAAGTGTACATTTGCGACCAAATGCGGAATCGCCTGAAAGATCGAGGTTTGGAGCTGATGGCCAAATGTTGCATTATGGAAGTGATGGATGCTTATGTTTATTCAACTGCTTCGCTTCTTGTTAATCAGTACCACGAATATGGAACGCTGCTTGAATATGCGAATAACATGAAGGATCCGAATTGGCACATAACCTGCTTCTTGATTACCCAAATGGCCCGAGTTGTGAAGGAAGTCCATGCCTCTAAAATTATTCATGGAGATATCAAACCGGATAATTTTATGATCACCAGAAAGTATGGGAAAACATTTGTTAATTTTAGACGTTATCTTTTTTCAGGATCGATGATAAATGGGGCAAAGATGCTCTGATGAGTAACGACAGCTTTGTCATCAAGATTATCAATTGGGGACGTGCGATTGACATGATGCCACTGAAGAACCAGCGTAACGCCAATCTAAATCCTAGAAGACGTCATThe introduced changes are shown in red, and synonymous mutations are shown in cyan.See C. elegans oocytes was used with minor modifications PVSEKTC. Serum was adsorbed with a non-phosphorylated peptide (RRRHLSPVSEKTC) followed by affinity purification with the antigenic, phosphorylated peptide. Different fractions were tested in immunofluorescence by incubating different dilutions  with 1 µM and 10 µM of either non-phosphorylated or phosphorylated peptide. Only the phosphorylated peptide compited out the signal. Additionally, we used the BUB-1d-lysine -coated slide, and a 24 × 24 cm coverslip was gently laid on top. Once the worms extruded the embryos, slides were placed on a metal block on dry ice for >10 min. The coverslip was then flicked off with a scalpel blade, and the samples were fixed in methanol at 20°C for 30 min. After blocking in phosphate-buffered saline (PBS) buffer plus 3% bovine serum albumin and 0.1% Triton X-100 (AbDil), samples were incubated overnight at 4°C with anti-BUB-1 . We used the strain HY604, which is a temperature-sensitive allele of the the APC component MAT-1, that arrests in meiosis I prior to spindle rotation when moved to the restrictive temperature.For GFP immunoprecipitations, we followed a published protocol  with minIP samples were run on 4–12% Bis–Tris sodium dodecyl sulfate gels with MOPS running buffer, and the gel was stained using Quick Coomassie Stain (Generon). Bands of interest were cut and washed with water:acetonitrile (50:50), followed by a wash with 100 mM ammonium bicarbonate. The gel pieces were then washed with 100 mM ammonium bicarbonate:acetonitrile (50:50), followed by a final wash with acetonitrile. Gel pieces were dried using a SpeedVac.Samples were reduced with 10 mM DTT in 20 mM ammonium bicarbonate and alkilated with 50 mM IAA (iodoacetamide) in 20 mM ammonium bicarbonate. Samples were then washed sequentially with 100 mM ammonium bicarbonate, 100 mM ammonium bicarbonate:acetonitrile (50:50), and acetonitrile. Gel pieces were dried using a SpeedVac.Trypsin solution (12.5 µg/ml stock in 20 mM ammonium bicarbonate) was added to cover the gel pieces and incubated for 30 min on a shaking platform and incubated sample overnight at 30°C on a shaker. Peptides were extracted by standard procedures and reconstituted in 10 µl of 5% formic acid/10% acetonitrile. After vortexing for 1 min, water was added to 50 µl.Samples were run on an Ultimate 3000 RSLCnano system (ThermoFisher Scientific) coupled with a Q-Exactive Plus Mass Spectrometer (Thermo Fisher Scientific). Peptides initially trapped on an Acclaim PepMap 100 (Thermo Fisher Scientific) and then separated on an Easy-Spray PepMap RSLC C18 column (Thermo Fisher Scientific). Sample was transferred to mass spectrometer via an Easy-Spray source with temperature set at 50°C and a source voltage of 2.1 kV. The mass spectrometer was operated on in data-dependent acquisition mode (top 15 method). MS resolution was 70,000 with a mass range of 350–1600. MS/MS resolution was 17,500.C. elegans proteome and BUB-1 specifically, using the Mascot Search Engine (Mascot Daemon Version 2.3.2). Type of search used was MS/MS Ion Search using Trypsin/P. Carbamidomethyl (C) was set as a fixed modification, and variable modifications were as follows: acetyl (N-term), dioxidation (M), Gln to pyro-Glu (N-term Q), oxidation (M), deamidation (NQ), and phosphorylation (STY).RAW data files were extracted and converted to mascot generic files (.mgf) using MSC Convert. Extracted data then searched against Peptide mass tolerance was ±10 ppm (# 13C = 2) with a fragment mass tolerance of ±0.6 Da. Maximum number of missed cleavages was 2.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner C. elegans PPTR-1 (UniProtKB O18178) and PPTR-2 (UniProtKB A9UJN4-1). A guide tree was calculated from the distance matrix generated from sequence pairwise scores.All sequence alignments were performed using Clustal Omega , versionC. elegans PPTR-1 (S420-V435) and PPTR-2 (S449-A464) were used for the alignment. The canonical sequences of each B56 isoform were retrieved from UniProt , δ (S454-A469), β (S409-V424), α (S403-V418), ε (S395-V410) and  UniProt .C. elegans BUB-1 , human BubR1 , and RepoMan  were aligned with Clustal Omega  or H1. At t = 0, 15 ng/µl Cdk1/Cyclin B was added, before incubation at 30°C for 30 min. Aliquots were taken immediately after Cdk1/CyclinB addition at t = 0 and then again after the 30 min incubation. All samples were then incubated at 70°C for 15 min in a final concentration of 1× LDS buffer. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was then conducted on a NuPage 4–12% Bis–Tris gel (Thermo) with MES buffer before being stained with ProQ Diamond (Thermo) and imaged using Bio-Rad ChemiDoc. Once fluorescence was recorded, Coomassie staining was performed. For the western blot, SDS–PAGE was conducted as above with 67 ng of substrate protein per well before the western was conducted using a nitrocellulose membrane  and 1× NuPage transfer buffer (Thermo). The membrane was blocked using Intercept PBS blocking buffer (LI-COR), the primary antibodies used were anti-GST at 1:1000 (made in sheep), and anti-phospho Ser 283 1:20,000 (made in rabbit). Secondary antibodies were anti-sheep IRDye 680RD anti-rabbit 800CW (LI-COR), both at 1:50,000. The membrane was then imaged using LI-COR Odyssey CLx.Forty microlitre reactions were set up containing 40 mM Tris–HCl pH 7.5, 100 µM ATP (Adenosine triphosphate), 10 mM MgClEscherichia coli strain BL21 (DE3) and the bacterial culture was incubated overnight at 37°C with shaking at 220 rpm. Bacteria were grown in TB medium at 37°C with shaking at 220 rpm until OD600 reached ~0.6–0.8 and induced with 150 μM IPTG. Induction was performed at 18°C with shaking at 220 rpm for ~16 hr. Cells were pelleted and lysed by sonication in 50 mM NaP, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 0.5 mM TCEP, (tris(2-carboxyethyl)phosphine) and protease inhibitors. After binding to a Ni-NTA column, protein was washed with 20 mM imidazole and then eluted with 350 mM imidazole. The tag was cleaved with 6xHis-tagged TEV (Tobacco Etch Virus Protease) protease overnight at 4°C, and the tag and protease were removed from the sample by binding to Ni-NTA. PPTR-2 was concentrated and further purified using a HiLoad 16/600 Superdex 200 pg size exclusion column.PPTR-2 was expressed from pHISTEV30a vector as a 6xHis- and Strep-tagged protein (plasmid fgp_445) in The following peptides were synthesised by peptides and elephants GmbH: BUB-1 FITC-Ahx-NPRRRHLSPVSEKTVDDEEE, pBUB-1 FITC-Ahx-NPRRRHLphSPVSEKTVDDEEE, and LAVA FITC-Ahx-NPRRRHASPASEKTVDDEEE. Reactions (35 µl) were set up in FP buffer  containing peptide concentrations 0.5–1 µM. These were then used to create a 1:2 serial dilution series using FP buffer containing the same peptide concentrations as above as well as the indicated concentration of PPTR-2. Reactions were left for 30 min before triplicates of each dilution were aliquoted into a black 384-well plate, centrifuged , and analysed using the PheraStar FS.For time-dependent analysis, metaphase I was taken as time = 0 s. This frame was chosen as the previous frame where the first indication of chromosome separation was visible. All image analysis was done in Fiji . For totCentral spindle to chromosome ratio for PPTR-2::GFP  was obtaFor the alignment/congression analysis, we selected videos in which the spindles were contained in a single Z-plane at −80 s. There, we established the spindle axes with a line extending from pole to pole and the ‘metaphase plate’ with a perpendicular line in the middle of the spindle see . A line Contingency tables were analysed using the Fisher’s exact test (two tailed), and the p values are presented in the figures and/or in the text.The 4D TIFF files were converted to video (.avi) files using a custom-made macro that uses the StackReg Fiji plugin for image registration. Videos were assembled using maximum-intensity projections; hence, the videos might not match a specific panel within the main figures, which are single slices or partial projections in order to highlight specific characteristics. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Using a combination of biochemistry, genetics and live imaging, Borja et al. show that the kinase BUB-1 recruits, through two regulatory subunits, the phosphatase PP2A to meiotic spindle chromosomes during oocyte meiosis I to promote proper congression of the chromosomes prior to anaphase. This recruitment occurs independently of the conserved protein Shugoshin, which has been shown by others to promote PP2A recruitment to chromosomes during mouse oocyte meiosis I. Moreover, Borja et al. show that phosphorylation of a peptide motif in BUB-1 promotes this recruitment, and that this BUB-1motif is likely targeted by CDK-1 for phosphorylation to provide proper temporal regulation of these events.Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]C. elegans Oocytes" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Jakob Nilsson (Reviewer #2); Patrick Meraldi (Reviewer #3).Thank you for submitting your work entitled "PP2A:B56 Regulates Meiotic Chromosome Segregation in eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife; in particular whether BUB1 directly binds to PP2A, through the LxxIxE motif, and whether Cdk phosphorylation of this is important. After discussion, they concluded that the amount of time and effort that will be required for these experiments is such that the fairest thing to do is to return the manuscript to you.You will see from the reviews that all the reviewers agree that your study will be of interest to the meiosis community in identifying that BUB1 rather than Sgo recruits PP2A, but that also all agree that more experiments are required to provide firm evidence for a number of your conclusions that would be necessary for publication in Reviewer #1:C. elegans oocyte meiotic cell division. They show that PP2A and two conserved B56-type regulatory subunits are localized to spindle poles, chromosomes and the central spindle (with some differences for the B56 subunits), and that all required for spindle assembly, chromosome congression to the metaphase plate, and for chromosome segregation (with some redundancy for the two B56 subunits). The further show that a conserved LxxIxE motif in BUB-1 recruits most of one and some of the other B56 subunit, and that the kinase domain of BUB-1 is required to recruit the other (presumably through an intermediary). Thus in C. elegans, a novel form of PP2A recruitment functions, with the other known recruiters, Shugoshin and Mad3 , appearing not be required. The authors use mutational analysis to nicely document the requirements for both parts of BUB-1 in PP2A recruitment, and also identify a phosphorylated residue in BUB-1 that may be involved in CDK-1 regulation of the recruitment. The authors propose that BUB-1 recruitment of PP2A to the central spindle is important for chromosome segregation during anaphase in C. elegans oocyte meiosis I.Borja et al. describe their analysis of the requirements for the phosphatase PP2A during C. elegans oocyte meiosis, with results that will be of substantial interest to investigators studyng oocyte meiotic cell division, the advances are in my opinion to incremental, and also lacking in conclusiveness as to the actual mechanism involved. Therefore as written the manuscript is not suitable for publication in eLife, as summarized in the major comments below.While the authors provide an extensive analysis of the requirements for PP2A during 1) The authors provide clear evidence that PP2A is required for spindle assembly, congression of chromosomes to the metaphase plate, and chromosome segregation (with some caveats as to chromosome segregation noted below). While the authors favor and propose that the chromosome segregation defects are due to central spindle defects , they in fact provide no evidence to support this conclusion. It is entirely possible that earlier spindle assembly defects are responsible for the subsequent defects in both congression and segregation. The authors fail to address this possibility and provide no evidence to rule it out. Without some idea as to which targets PP2A acts through, the advance is incremental relative to what is known in other systems, only showing a variation on how LxxIxE motifs can recruit PP2A to spindle structures through BUB-1 instead of other factors. The authors discuss the AuroraB kinase AIR-2 as a possible target but provide no analysis of such a role. Without more mechanistic insight, the manuscript is of substantial interest but seems more appropriate for a more specialized journal such as Molecular Biology of the Cell.C. elegans mutants that chromosomes segregation and polar extrusion are not tightly correlated. The authors should establish a more direct approach to quantifying the defects in chromosome segregation; as written only representative examples are shown.2) In figures throughout the manuscript, the authors use polar body extrusion as a proxy for chromosome segregation defects. However, it is known from work on other 3) The authors show that there are severe spindle assembly defects after knockdown of PP2A but only show microtubules and chromosomes to document the defects. Given that the spindle assembly defects might be primarily responsible for subsequent defects in chromosome alignment and segregation, the authors should better characterize the spindle assembly defects using pole marker(s). Do mutant oocytes even establish a bipolar spindle? From the images, it seems likely they do not.4) The authors generate point mutations in both the LxxIxE motif and the kinase domain of BUB-1 to nicely document requirements for recruiting PP2A. However, the authors do not include any mention of whether these mutations are essential for oocyte meiotic cell division, or if the mutations are associated with any embryonic lethality . In fact, from the supplemental tables it appears that both mutant strains are homozygous viable, although this is never mentioned in the text. If these motifs are indeed responsible for recruiting PP2A and its essential functions to the spindle, then the mutants should result in defects identical to the PP2A knockdowns. Thus to verify the importance of these mutations, the authors would need to construct a balanced strain in which both motifs are mutated and document defects much like those observed in the PP2A knockdowns. More generally, the authors should provide a genetic analysis of the viability of the single mutants .Reviewer #2:C. elegans. Although the shugoshin proteins have been shown to be important regulators of cohesin during meiosis by recruiting PP2A-B56 in other systems the authors show this is not the case in C. elegans. Instead they show that the Bub1 protein recruits PP2A-B56 through a LxxIxE motif that resembles the one found in human BubR1. Mutation of this motif prevents the recruitment of the PPTR1 (one of the B56 isoforms) to the midbivalent and the central spindle and reduces the recruitment of the PPTR2 to the central spindle. The Bub1 variant with a mutated LxxIxE motif displays misalignments suggesting an imbalance in phosphorylations. The phenotype of the Bub1 mutant is not as severe as Bub1 RNAi but this could be because the kinase domain of Bub1 helps in recruiting PPTR2.This manuscript explores female meiosis and the role of Bub1 in targeting PP2A-B56 to chromosomes and central spindle for proper meiosis in eLife.Overall the paper is easy to read and the data are consistent. However, the paper is fairly descriptive and lacks to some degree mechanistic insight. I think that some additional experiments would make the work more interesting for the readers of 1) Characterization of the double Bub1 mutant with a mutated LxxIxE motif and mutations in the kinase domain. This would clarify if there is some redundancy in recruitment mechanisms2) Direct evidence that the LxxIxE motif of Bub1 facilitates binding to PPTR1/2 – IP or purified components3) Characterization of the LAPI mutation of the LxxIxE motif to determine if Cdk1 phosphorylation of this site is important. Reading the manuscript as it is now this is purely speculative despite they go through the effort of showing phosphorylation.Reviewer #3:C. elegans anaphase. It finds that PP2A plays a crucial role in chromosome alignment and segregation. This role depends on the B56 regulatory subunits. Moreover, the author show that the recruitment of the B56 subunits to the spindle apparatus and kinetochores in anaphase depends on the Bub-1 kinase.The study by Bel Borja and colleagues studies to which extent the PP2A phosphatase contributes to chromosome segregation in C. elegans anaphase, and is generally of good technical quality (see below), yet at the same time feels a bit thin in terms of results. In particular several major claims of the discussion are not supported by the data. Moreover, it is not clear to which extent the reported experiments are reproducible.The study explores a novel role of PP2A in 1) The authors do not indicate the number of independent experiments or the number of embryos on which the conclusions are based on. This information is essential to evaluate the reproducibility of the results. The authors use good statistical tests, but the reader must know whether the experiments are based on 5, 10 or 20 embryos.C. elegans anaphase by RECRUITING PP2A:B56 to the spindle apparatus. The presented experiments only show that the localization of the B56 depends on Bub-1, which is not the same thing. The authors could test by IP and in vitro pull-down that the claimed interaction is direct (in vitro), or that the proteins are part of the same complex (in vivo). This should be feasible since the authors already have performed IP experiments.2) The authors claim the Bub-1 acts on 3) The authors claim that PP2A:B56 counteracts Aurora-B during anaphase, but have no evidence for this claim. Does a Aurora-B hypomorph mutant attenuate the B56 loss phenotype? While a negative result would not mean that the model is wrong, a positive result would certainly help to bolster that claim. [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]You will see from the reviews that all the reviewers agree that your study will be of interest to the meiosis community in identifying that BUB1 rather than Sgo recruits PP2A, but that also all agree that more experiments are required to provide firm evidence for a number of your conclusions that would be necessary for publication in eLife; in particular whether BUB1 directly binds to PP2A, through the LxxIxE motif, and whether Cdk phosphorylation of this is important. After discussion, they concluded that the amount of time and effort that will be required for these experiments is such that the fairest thing to do is to return the manuscript to you.The new version of the manuscript has numerous new experiments to address reviewers’ comments/concerns. Relating to the particular two points highlighted by the reviewing editor, we provide data showing that the BUB-1 LxxIxE motif binds the B56 orthologue PPTR-2 in vitro and Ser 283 phosphorylation increases affinity. Furthermore, mutating Ser 283 to Ala drastically inhibits B56 recruitment in vivo resulting in a chromosome alignment defect similar to the L282A,V285A mutant.We also performed in vitro kinase assays and showed that Cdk1 can phosphorylate Ser 283 in BUB-1 and developed a phospho-specific antibody recognising phosphor-Ser 283 and show that phosphorylated BUB-1 is detected … in vivo.Thanks to the new quantitative phenotypic analysis we performed, it became clear that B56 subunits play a role during chromosome alignment/congression prior to anaphase. Furthermore, this relies on B56 recruitment by the newly identified BUB-1 LxxIxE motif.Reviewer #1:[…]1) The authors provide clear evidence that PP2A is required for spindle assembly, congression of chromosomes to the metaphase plate, and chromosome segregation (with some caveats as to chromosome segregation noted below). While the authors favor and propose that the chromosome segregation defects are due to central spindle defects , they in fact provide no evidence to support this conclusion. It is entirely possible that earlier spindle assembly defects are responsible for the subsequent defects in both congression and segregation. The authors fail to address this possibility and provide no evidence to rule it out.We agree we might have oversimplified the phenotypes and the connection between them. Our biggest mistake was to base the alignment analysis on subjective observations its classification as either “aligned” or “misaligned”.Firstly, we performed a new analysis related to the chromosome alignment phenotype. This is now detailed in the Materials and methods section and it provides more rigorous and less subjective quantifications which lead us to a better characterisation of this phenotype. This had a tremendous impact on the manuscript, because it is precisely this process of chromosome alignment where BUB-1 targeted PP2A:B56 plays a role. To avoid any potential amplification of the phenotypes due to partial loss of function in PP2A subunits, we decided to measure the phenotypes on the GFP::tubulin expressing strain and not on the strains with tagged PP2A subunits.PP2A is essential for the assembly of a bipolar spindle  and chromosome alignment and segregation. While we agree with the reviewer that in this case it is difficult to tease out whether the alignment and segregation are only a direct consequence of this, depletion of the B56 subunits PPTR-1 and PPTR-2 does not affect spindle assembly but perturbs chromosome alignment/congression . Since this phenotype is measurable and independent from any noticeable spindle defect, we focused on this “clean” phenotype and sought to understand the molecular mechanisms driving it.Without some idea as to which targets PP2A acts through, the advance is incremental relative to what is known in other systems, only showing a variation on how LxxIxE motifs can recruit PP2A to spindle structures through BUB-1 instead of other factors. The authors discuss the AuroraB kinase AIR-2 as a possible target but provide no analysis of such a role. Without more mechanistic insight, the manuscript is of substantial interest but seems more appropriate for a more specialized journal such as Molecular Biology of the Cell.C. elegans, it was not clear what role(s) BUB-1 play. Therefore, we think we are providing a significant advance on the mechanisms in place to regulate meiosis I. While we are actively working on identifying PP2A substrates and its potential role in antagonising Aurora B, we think this will require an in depth analysis that will be the focus of another paper.While we respect the reviewer’s view on the advance provided by our manuscript, we do believe we provide mechanistic insight into the targeting of PP2A:B56 in a context in which the previously known/characterised regulators (BubR1 and Shugoshin) do not play a role. On the other hand, while BUB-1 is known to be important during female meiosis in C. elegans mutants that chromosomes segregation and polar extrusion are not tightly correlated. The authors should establish a more direct approach to quantifying the defects in chromosome segregation; as written only representative examples are shown.2) In figures throughout the manuscript, the authors use polar body extrusion as a proxy for chromosome segregation defects. However, it is known from work on other We apologise for the misunderstanding. We did not intend to use PB extrusion as a proxy for chromosome segregation defects, but rather analysed it as an independent process. We are now making this clear in the manuscript. We analyse chromosome alignment defects (see below), lagging chromosomes, and polar body extrusion. For these three phenomena, we provide the quantitative assessment for each condition and show a representative image (or sets of images).3) The authors show that there are severe spindle assembly defects after knockdown of PP2A but only show microtubules and chromosomes to document the defects. Given that the spindle assembly defects might be primarily responsible for subsequent defects in chromosome alignment and segregation, the authors should better characterize the spindle assembly defects using pole marker(s). Do mutant oocytes even establish a bipolar spindle? From the images, it seems likely they do not.As requested by the reviewer, we have performed new experiments using GFP-ASPM-1 as a pole marker. As expected, LET-92-depleted oocytes fail to assemble a bipolar spindle . As mentioned above, in order to differentiate subsequent phenotypes from the spindle defect, we focused on the alignment/congression defects which are observed even in the presence of a seemingly normal bipolar spindle.4) The authors generate point mutations in both the LxxIxE motif and the kinase domain of BUB-1 to nicely document requirements for recruiting PP2A. However, the authors do not include any mention of whether these mutations are essential for oocyte meiotic cell division, or if the mutations are associated with any embryonic lethality . In fact, from the supplemental tables it appears that both mutant strains are homozygous viable, although this is never mentioned in the text. If these motifs are indeed responsible for recruiting PP2A and its essential functions to the spindle, then the mutants should result in defects identical to the PP2A knockdowns. Thus to verify the importance of these mutations, the authors would need to construct a balanced strain in which both motifs are mutated and document defects much like those observed in the PP2A knockdowns. More generally, the authors should provide a genetic analysis of the viability of the single mutants .Full analysis of embryo viability and brood size analysis is now presented for all the mutants used in the study .Reviewer #2:C. elegans. Although the shugoshin proteins have been shown to be important regulators of cohesin during meiosis by recruiting PP2A-B56 in other systems the authors show this is not the case in C. elegans. Instead they show that the Bub1 protein recruits PP2A-B56 through a LxxIxE motif that resembles the one found in human BubR1. Mutation of this motif prevents the recruitment of the PPTR1 (one of the B56 isoforms) to the midbivalent and the central spindle and reduces the recruitment of the PPTR2 to the central spindle. The Bub1 variant with a mutated LxxIxE motif displays misalignments suggesting an imbalance in phosphorylations. The phenotype of the Bub1 mutant is not as severe as Bub1 RNAi but this could be because the kinase domain of Bub1 helps in recruiting PPTR2.This manuscript explores female meiosis and the role of Bub1 in targeting PP2A-B56 to chromosomes and central spindle for proper meiosis in Overall the paper is easy to read and the data are consistent. However, the paper is fairly descriptive and lacks to some degree mechanistic insight. I think that some additional experiments would make the work more interesting for the readers of eLife.1) Characterization of the double Bub1 mutant with a mutated LxxIxE motif and mutations in the kinase domain. This would clarify if there is some redundancy in recruitment mechanisms.We have performed the suggested experiment. The results indicate that LxxIxE motif-mediated recruitment of B56 subunits operates mostly in the midbivalent and central spindle, whereas the kinase domain mediates mostly chromosome chromosomal recruitment of the B56 subunits .2) Direct evidence that the LxxIxE motif of Bub1 facilitates binding to PPTR1/2 – IP or purified components.We have performed in vitro binding experiments and present evidence for the direct interaction between the LxxIxE motif of BUB-1 and B56. We expressed recombinant, full-length PPTR-2 in bacteria and performed fluorescence polarisation experiments using fluorescently labelled LxxIxE motif peptides. We could observe that PPTR-2 bound the wild - type sequence and that L282A,V285A mutations abolished binding . On the contrary, binding was enhanced when the peptide was phosphorylated at Serine 283 .3) Characterization of the LAPI mutation of the LxxIxE motif to determine if Cdk1 phosphorylation of this site is important. Reading the manuscript as it is now this is purely speculative despite they go through the effort of showing phosphorylation.We have now expanded on the LxxIxE motif phosphorylation and its role in vivo.1) in vitro assays demonstrate that Cdk1 phosphorylates Serine 283 in vitro. This is now part of the New Figure 5C-E.S283A, achieving a similar effect to that of BUB-1L282A,V285A. Previous data showed that substituting I for V in the LxxIxE motif decreases affinity and we believe in this case this renders the motif more dependent on phosphorylation. These results are included in the New Figure 6.2) To analyse the role of Serine 283 phosphorylation in vivo, we mutated it to alanine in endogenous BUB-1. PPTR-1 and PPTR-2 localisation to the midbivalent and central spindle was significantly affected in BUB-13) In addition to the previously reported mass spec data, we now show the localisation of the L(pS)PVSE motif by immunofluorescence with a newly generated phospho-specific antibody .Reviewer #3:C. elegans anaphase. It finds that PP2A plays a crucial role in chromosome alignment and segregation. This role depends on the B56 regulatory subunits. Moreover, the author show that the recruitment of the B56 subunits to the spindle apparatus and kinetochores in anaphase depends on the Bub-1 kinase.The study by Bel Borja and colleagues studies to which extent the PP2A phosphatase contributes to chromosome segregation in C. elegans anaphase, and is generally of good technical quality (see below), yet at the same time feels a bit thin in terms of results. In particular several major claims of the discussion are not supported by the data. Moreover, it is not clear to which extent the reported experiments are reproducible.The study explores a novel role of PP2A in 1) The authors do not indicate the number of independent experiments or the number of embryos on which the conclusions are based on. This information is essential to evaluate the reproducibility of the results. The authors use good statistical tests, but the reader must know whether the experiments are based on 5, 10 or 20 embryos.We have now included all the information in the graphs. For intensity measurements, “N” is the number of experiments, and “n” is the number of oocytes. For the congression and alignment analysis, “N” is the number of spindles (=number of oocytes) and “n” is the number of bivalents analysed. In the latter analysis, information on the number of experiments used to obtain the data is stated in the figure legend.C. elegans anaphase by RECRUITING PP2A:B56 to the spindle apparatus. The presented experiments only show that the localization of the B56 depends on Bub-1, which is not the same thing. The authors could test by IP and in vitro pull-down that the claimed interaction is direct (in vitro), or that the proteins are part of the same complex (in vivo). This should be feasible since the authors already have performed IP experiments.2) The authors claim the Bub-1 acts on We thank the reviewer for raising this concern and we have now changed the wording when referring to this. We have performed in vitro binding experiments and present evidence for the direct interaction between the LxxIxE motif of BUB-1 and PPTR-2 . We expressed recombinant, full-length PPTR-2 in bacteria and performed fluorescence polarisation experiments using fluorescently labelled LxxIxE motif peptides. PPTR-2 bound the wild type sequence and that binding was abolished by the L282A,V285A mutations and enhanced when the peptide was phosphorylated at Serine 283.3) The authors claim that PP2A:B56 counteracts Aurora-B during anaphase, but have no evidence for this claim. Does a Aurora-B hypomorph mutant attenuate the B56 loss phenotype? While a negative result would not mean that the model is wrong, a positive result would certainly help to bolster that claim.As stated above, we think we are providing a significant advance on the mechanisms in place to regulate meiosis I by specific recruitment of PP2A/B56. While we are actively working on identifying PP2A substrates and its potential role in antagonising Aurora B, we believe this will require an in-depth analysis that will be the focus of another paper. We have now focused our discussion on our actual data and left this as an interesting, yet unsupported, hypothesis.


To assess the role of a protein, protein loss phenotypic studies can be used, most commonly through mutagenesis RNAi or CRISPR knockout. Such studies have been critical for the understanding of protein function and the identification of putative therapeutic targets for numerous human disease states. However, these methodological approaches present challenges because they are not easily reversible, and if an essential gene is targeted, an associated loss of cell viability can potentially hinder further studies. Here we present a reversible and conditional live‐cell knockout strategy that is applicable to numerous proteins. This modular protein‐tagging approach regulates target loss at the protein, rather than the genomic, level through the use of HaloPROTAC3, which specifically degrades HaloTag fusion proteins via recruitment of the VHL E3 ligase component. To enable HaloTag‐mediated degradation of endogenous proteins, we provide protocols for HaloTag genomic insertion at the protein N or C terminus via CRISPR/Cas9 and use of HaloTag fluorescent ligands to enrich edited cells via Fluorescence‐Activated Cell Sorting (FACS). Using these approaches, endogenous HaloTag fusion proteins present in various subcellular locations can be degraded by HaloPROTAC3. As detecting the degradation of endogenous targets is challenging, the 11‐amino‐acid peptide tag HiBiT is added to the HaloTag fusion to allows the sensitive luminescence detection of HaloTag fusion levels without the use of antibodies. Lastly, we demonstrate, through comparison of HaloPROTAC3 degradation with that of another fusion tag PROTAC, dTAG‐13, that HaloPROTAC3 has a faster degradation rate and similar extent of degradation. © 2020 The Authors.Basic Protocol 1: CRISPR/Cas9 insertion of HaloTag or HiBiT‐HaloTagBasic Protocol 2: HaloPROTAC3 degradation of endogenous HaloTag fusions Targeted protein degradation using proteolysis targeting chimeras (PROTACs) is a rapidly growing research area and an exciting new modality of therapeutic treatment  donor vector. After CRISPR pools are generated, further details are provided for enrichment of HaloTag positive cells using the HaloTag fluorophore, Janelia Fluor 646 (JF646) HaloTag ligand  be placed immediately following the endogenous target start codons or any type of signal sequence that could be cleaved. If introducing HaloTag or HaloTag‐HiBiT at the C terminus, place the tag(s) immediately upstream of the native stop codon. Generation of genomic maps that include the CRISPR insertions will aid in ensuring the tags are placed in the proper reading frame. As CRISPR insertion success can be highly dependent upon the choice of guide RNA, it is advisable to test a minimum of two different guide RNA sequences. Initial studies appending the tag(s) on the N‐ or C‐terminal end of the protein using expression vectors can be used to determine the tag(s) effects on the protein's expression, binding interactions, and folding abilities. However, overexpression is not recommended for degradation studies with HaloPROTAC3, as little degradation will be detected due to the high expression levels of the fusion protein.Alt‐R CRISPR‐Cas9 tracrRNA Alt‐R CRISPR‐Cas9 CRISPR RNA Nuclease Free Duplex Buffer Alt‐R S.p. Cas9 Nuclease Desired cell line for CRISPR modification that is amenable to nucleofectionDPBS 0.05% trypsin/EDTA  or 0.25% trypsin/EDTA , depending on cell lineComplete growth medium for cell type of choiceMirus Ingenio Solution HaloTag or HiBiT‐HaloTag double‐stranded (ds) DNA donor plasmid Janelia Fluor 646 (JF646) HaloTag Ligand 100× Antibiotic/Antimycotic solution FACS buffer see Optional, for use of HiBiT‐HaloTag or HaloTag‐HiBiT CRISPR insertions: Nano‐GloHiBiT Lytic Detection System Heat block 50‐ml conical tubes Mirus Ingenio Kit Bio‐Rad Gene Pulser Xcell Electroporation SystemAppropriate incubator for cell lineClear, tissue‐culture‐grade 96‐well plates 5‐ml round‐bottom tube with cell strainer cap BD FACS Melody or similarWhite 96‐well plates Optional, for use of HiBiT‐HaloTag or HaloTag‐HiBiT CRISPR insertions: Luminometer such as the GloMax Discover Microplate Reader  or CLARIOstar Plus (BMG Labtech)Additional reagents and equipment for basic cell culture techniques, including cell counting  complex by combining 75 pmol Cas9 and 120 pmol tracrRNA:crRNA duplex:Add the Cas9 very slowly to avoid precipitation, and swirl with pipet tip.5Incubate RNP complex 10‐20 min at room temperature.66 cells/reaction), remove cells from plasticware using appropriate splitting techniques, centrifuge cells at 200 × g for 5 min, rinse cells with DPBS, and centrifuge again. For example, if using HEK293 cells, wash with DPBS, detach cells with trypsin, inactivate trypsin with DMEM + 10% FBS, and transfer cells to a conical tube for centrifugation.Determine total number of cells needed based on number of CRISPR reactions  for the cell type, and split cells if they become confluent. If performing HiBiT‐HaloTag CRISPR insertions, HiBiT lytic luminescence assays can be performed to determine insertion efficiency  in triplicate to a white 96‐well assay plate.29Dilute the LgBiT Protein 1:100 and the Nano‐Glo HiBiT Lytic Substrate 1:50 into an appropriate volume of room‐temperature Nano‐Glo HiBiT Lytic Buffer in a new tube. Mix by inversion.30Add 100 µl Nano‐Glo HiBiT Lytic Reagent to each well.31Mix the samples by placing the plate on an orbital shaker (300‐600 rpm) for at least 10 min.32Read luminescence with GloMax Discover Microplate Reader using a 0.5‐s integration time Fig. .Basic Protocol 2ent‐HaloPROTAC3  is an alternate procedure for the live‐cell kinetic degradation analysis of HiBiT‐HaloTag insertions to better understand degradation rate, optimal time of treatments, and kinetic dose‐response curves. The use of HiBiT for protein level detection is highly quantitative, directly correlative to the endogenous target protein level, and does not require the use of antibodies 0.05% trypsin/EDTA  or 0.25% trypsin/EDTA , depending on cell lineComplete growth medium for cell type of choiceHaloPROTAC3 ligand ent‐HaloPROTAC3 ligand Dimethyl sulfoxide Optional materials for live‐cell kinetic degradation of HiBiT‐HaloTag fusions:LgBiT Expression Vector Nano‐Glo Endurazine Live Cell Substrate 2‐independent medium COFBS Six‐well platesAppropriate incubator for cell lineDilution reservoirs White 96‐well plates Optional material for live‐cell kinetic degradation of HiBiT‐HaloTag fusions: Luminometer such as the GloMax Discover Microplate Reader  or CLARIOstar Plus (BMG Labtech)1Culture CRISPR/Cas9‐edited cells carrying HaloTag, HiBiT‐HaloTag , or HaloTag‐HiBiT  insertions appropriately in preparation for the assay.If performing live‐cell degradation assays with HiBiT‐HaloTag CRISPR insertions proceed to optional live‐cell HiBiT‐HaloTag degradation assay at the end of Basic Protocol 2For adherent cells, wash cells with DPBS and trypsinize.3ent‐HaloPRTOAC3, and one for DMSO control.Count cells and plate 800,000 cells per well in a six‐well plate. Plate one well for HaloPROTAC3, one for 4For adherent cells, incubate cells overnight (18‐24 hr) in an appropriate incubator. For suspension cells, proceed to step 5.5ent‐HaloPROTAC3, and (c) a volume of DMSO equivalent to that added in the PROTAC treatments.Add ligands to growth medium in well to obtain final concentrations of (a) 300 nM HaloPROTAC3, (b) 300 nM For example, if there is 2 ml of growth medium in well, add 500 µl 1.5 µM HaloPROTAC3 stock to make a solution of 300 nM final concentration.6Incubate cells in an appropriate incubator overnight.These recommended HaloPROTAC3 concentrations and treatment times can be increased or decreased depending upon target and desired level of degradation.7Detect degradation of endogenous HaloTag target fusion. If using HiBiT‐HaloTag CRISPR insertions, HiBiT lytic luminescence assays can be carried out to quantitate degradation following Basic Protocol 8Transfect the LgBiT vector into HaloTag‐ or HiBiT‐HaloTag‐edited cells using standard transient transfection and following manufacturer's recommendations.9Incubate plates in an appropriate incubator overnight (18‐24 hr).102‐independent medium with the appropriate percentage of FBS for the cell type, or proper assay medium for cells, by diluting the stock reagent of the substrate 1:100. If the luminometer provides CO2 injection or cells do not require CO2, regular assay medium can be used.Prepare a 1× solution of Nano‐Glo Endurazine Live Cell Substrate in CO11Aspirate cell culture medium from plate, and add 90 µl of the Endurazine solution to each well.122 level (if possible or needed) of cell line in use.Incubate plate for at least 2.5 hr at in an incubator set to the appropriate growing conditions for the cells to equilibrate the luminescence. During this incubation period, it is advisable to pre‐equilibrate the luminometer to the growth temperature and CO13ent‐HaloPROTAC3 either in CO2‐independent medium containing the appropriate percentage of FBS for the cell type or in proper assay medium for the cells.Prepare 10 µM solutions of HaloPROTAC3 and 14ent‐HaloPROTAC3 in dilution reservoirs using assay medium that contains DMSO at the same concentration as the 10 µM stock. Reserve the last well of the dilution series to contain DMSO alone, as a negative control. This results in a 10× dilution series, which will be further diluted upon addition to cells for a final treatment concentration of 1 µM at the highest point.Perform threefold dilutions of 10 µM HaloPROTAC3 or 15ent‐HaloPROTAC3 ligand to each well of the 96‐well plate.Add 10 µl of the diluted HaloPROTAC3 or 16Immediately place the plate in a luminometer plate reader pre‐equilibrated to the growth temperature of the cell line in use.17Read luminescence at desired intervals. A recommended starting point is every 15 min over a 24‐hr total time frame.18ent‐HaloPROTAC3‐treated wells by those from the DMSO control . Examples of kinetic degradation profiles of HiBiT‐HaloTag CRISPR insertions are shown in Figures Calculate the fractional RLUs at each time point by dividing the RLU value from HaloPROTAC3‐treated or 1× Hank's Balanced Salt Solution 10 mM HEPES 0.2% BSA 1× Antibiotic/Antimycotic Solution N‐terminal HaloTag sequence:500‐bp upstream homology arm‐GCAGAAATCGGTACTGGCTTTCCATTCGACCCCCATTATGTGGAAGTCCTGGGCGAGCGCATGCACTACGTCGATGTTGGTCCGCGCGATGGCACCCCTGTGCTGTTCCTGCACGGTAACCCGACCTCCTCCTACGTGTGGCGCAACATCATCCCGCATGTTGCACCGACCCATCGCTGCATTGCTCCAGACCTGATCGGTATGGGCAAATCCGACAAACCAGACCTGGGTTATTTCTTCGACGACCACGTCCGCTTCATGGATGCCTTCATCGAAGCCCTGGGTCTGGAAGAGGTCGTCCTGGTCATTCACGACTGGGGCTCCGCTCTGGGTTTCCACTGGGCCAAGCGCAATCCAGAGCGCGTCAAAGGTATTGCATTTATGGAGTTCATCCGCCCTATCCCGACCTGGGACGAATGGCCAGAATTTGCCCGCGAGACCTTCCAGGCCTTCCGCACCACCGACGTCGGCCGCAAGCTGATCATCGATCAGAACGTTTTTATCGAGGGTACGCTGCCGATGGGTGTCGTCCGCCCGCTGACTGAAGTCGAGATGGACCATTACCGCGAGCCGTTCCTGAATCCTGTTGACCGCGAGCCACTGTGGCGCTTCCCAAACGAGCTGCCAATCGCCGGTGAGCCAGCGAACATCGTCGCGCTGGTCGAAGAATACATGGACTGGCTGCACCAGTCCCCTGTCCCGAAGCTGCTGTTCTGGGGCACCCCAGGCGTTCTGATCCCACCGGCCGAAGCCGCTCGCCTGGCCAAAAGCCTGCCTAACTGCAAGGCTGTGGACATCGGCCCGGGTCTGAATCTGCTGCAAGAAGACAACCCGGACCTGATCGGCAGCGAGATCGCGCGCTGGCTGTCGACGCTCGAGATTTCCGGCGAGCCAACCACTGAGGATCTGTACTTTCAGAGCGATAAC‐500‐bp downstream homology armC‐terminal HaloTag sequence:500‐bp upstream homology arm‐GAGCCAACCACTGAGGATCTGTACTTTCAGAGCGATAACGATGGATCCGAAATCGGTACTGGCTTTCCATTCGACCCCCATTATGTGGAAGTCCTGGGCGAGCGCATGCACTACGTCGATGTTGGTCCGCGCGATGGCACCCCTGTGCTGTTCCTGCACGGTAACCCGACCTCCTCCTACGTGTGGCGCAACATCATCCCGCATGTTGCACCGACCCATCGCTGCATTGCTCCAGACCTGATCGGTATGGGCAAATCCGACAAACCAGACCTGGGTTATTTCTTCGACGACCACGTCCGCTTCATGGATGCCTTCATCGAAGCCCTGGGTCTGGAAGAGGTCGTCCTGGTCATTCACGACTGGGGCTCCGCTCTGGGTTTCCACTGGGCCAAGCGCAATCCAGAGCGCGTCAAAGGTATTGCATTTATGGAGTTCATCCGCCCTATCCCGACCTGGGACGAATGGCCAGAATTTGCCCGCGAGACCTTCCAGGCCTTCCGCACCACCGACGTCGGCCGCAAGCTGATCATCGATCAGAACGTTTTTATCGAGGGTACGCTGCCGATGGGTGTCGTCCGCCCGCTGACTGAAGTCGAGATGGACCATTACCGCGAGCCGTTCCTGAATCCTGTTGACCGCGAGCCACTGTGGCGCTTCCCAAACGAGCTGCCAATCGCCGGTGAGCCAGCGAACATCGTCGCGCTGGTCGAAGAATACATGGACTGGCTGCACCAGTCCCCTGTCCCGAAGCTGCTGTTCTGGGGCACCCCAGGCGTTCTGATCCCACCGGCCGAAGCCGCTCGCCTGGCCAAAAGCCTGCCTAACTGCAAGGCTGTGGACATCGGCCCGGGTCTGAATCTGCTGCAAGAAGACAACCCGGACCTGATCGGCAGCGAGATCGCGCGCTGGCTGTCTACTCTGGAGATTTCCGGT‐500‐bp downstream homology armN‐terminal HiBiT‐HaloTag sequence:500‐bp upstream homology arm‐GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGCAGAAATCGGTACTGGCTTTCCATTCGACCCCCATTATGTGGAAGTCCTGGGCGAGCGCATGCACTACGTCGATGTTGGTCCGCGCGATGGCACCCCTGTGCTGTTCCTGCACGGTAACCCGACCTCCTCCTACGTGTGGCGCAACATCATCCCGCATGTTGCACCGACCCATCGCTGCATTGCTCCAGACCTGATCGGTATGGGCAAATCCGACAAACCAGACCTGGGTTATTTCTTCGACGACCACGTCCGCTTCATGGATGCCTTCATCGAAGCCCTGGGTCTGGAAGAGGTCGTCCTGGTCATTCACGACTGGGGCTCCGCTCTGGGTTTCCACTGGGCCAAGCGCAATCCAGAGCGCGTCAAAGGTATTGCATTTATGGAGTTCATCCGCCCTATCCCGACCTGGGACGAATGGCCAGAATTTGCCCGCGAGACCTTCCAGGCCTTCCGCACCACCGACGTCGGCCGCAAGCTGATCATCGATCAGAACGTTTTTATCGAGGGTACGCTGCCGATGGGTGTCGTCCGCCCGCTGACTGAAGTCGAGATGGACCATTACCGCGAGCCGTTCCTGAATCCTGTTGACCGCGAGCCACTGTGGCGCTTCCCAAACGAGCTGCCAATCGCCGGTGAGCCAGCGAACATCGTCGCGCTGGTCGAAGAATACATGGACTGGCTGCACCAGTCCCCTGTCCCGAAGCTGCTGTTCTGGGGCACCCCAGGCGTTCTGATCCCACCGGCCGAAGCCGCTCGCCTGGCCAAAAGCCTGCCTAACTGCAAGGCTGTGGACATCGGCCCGGGTCTGAATCTGCTGCAAGAAGACAACCCGGACCTGATCGGCAGCGAGATCGCGCGCTGGCTGTCGACGCTCGAGATTTCCGGCGAGCCAACCACTGAGGATCTGTACTTTCAGAGCGATAAC‐500‐bp downstream homology armC‐terminal HaloTag‐VS‐HiBiT sequence:500‐bp upstream homology arm‐ GAGCCAACCACTGAGGATCTGTACTTTCAGAGCGATAACGATGGATCCGAAATCGGTACTGGCTTTCCATTCGACCCCCATTATGTGGAAGTCCTGGGCGAGCGCATGCACTACGTCGATGTTGGTCCGCGCGATGGCACCCCTGTGCTGTTCCTGCACGGTAACCCGACCTCCTCCTACGTGTGGCGCAACATCATCCCGCATGTTGCACCGACCCATCGCTGCATTGCTCCAGACCTGATCGGTATGGGCAAATCCGACAAACCAGACCTGGGTTATTTCTTCGACGACCACGTCCGCTTCATGGATGCCTTCATCGAAGCCCTGGGTCTGGAAGAGGTCGTCCTGGTCATTCACGACTGGGGCTCCGCTCTGGGTTTCCACTGGGCCAAGCGCAATCCAGAGCGCGTCAAAGGTATTGCATTTATGGAGTTCATCCGCCCTATCCCGACCTGGGACGAATGGCCAGAATTTGCCCGCGAGACCTTCCAGGCCTTCCGCACCACCGACGTCGGCCGCAAGCTGATCATCGATCAGAACGTTTTTATCGAGGGTACGCTGCCGATGGGTGTCGTCCGCCCGCTGACTGAAGTCGAGATGGACCATTACCGCGAGCCGTTCCTGAATCCTGTTGACCGCGAGCCACTGTGGCGCTTCCCAAACGAGCTGCCAATCGCCGGTGAGCCAGCGAACATCGTCGCGCTGGTCGAAGAATACATGGACTGGCTGCACCAGTCCCCTGTCCCGAAGCTGCTGTTCTGGGGCACCCCAGGCGTTCTGATCCCACCGGCCGAAGCCGCTCGCCTGGCCAAAAGCCTGCCTAACTGCAAGGCTGTGGACATCGGCCCGGGTCTGAATCTGCTGCAAGAAGACAACCCGGACCTGATCGGCAGCGAGATCGCGCGCTGGCTGTCTACTCTGGAGATTTCCGGTGTCTCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC‐500‐bp downstream homology armKey: blue, HiBiT; red, HaloTag; green, linker.Targeted protein degradation has resulted in an explosion of new avenues of research, from therapeutic drug discovery and clinical trials to the expansion of E3 ligase studies and phenotypic studies  as compared to smaller insertions, for which single‐stranded oligodeoxynucleotide (ssODN) synthesis is possible and homology arms (30‐50bp each) are shorter. The use of dsDNA donor vectors in general results in low insertion efficiency, and expected pool percentage of edited cells with this approach could range between 0.1% and 15%. For insertion of HaloTag or HiBiT‐HaloTag, the use of donor vector without a promoter is important to promote specific, on‐target insertion and minimize random integration of vector, which could then result in expression of the tag alone. To identify CRISPR HaloTag target‐edited cells from random integration, it is important to assess that the proper‐sized fusion is made by amplifying the genomic region by PCR or visualizing protein size on a protein gel with the HaloTag TMR ligand  on untagged alleles, resulting in a knockout of the untagged protein copies. As a result, the entire target protein pool in these clones is expressed as a fusion to HaloTag; therefore, these clones are sufficient for phenotypic experiments with HaloPROTAC3.HaloPROTAC3 will degrade HaloTag target fusion proteins that are recruited to the VHL E3 ligase component, incorporated into active E2/E3 ligase complexes for ubiquitination, and then trafficked to the proteasome. VHL is expressed throughout the cytoplasm and nucleus, as well as in numerous cell types (Buckley et al., As endogenous target proteins have highly variable expression levels that will not be significantly altered by HaloTag insertion, varying concentrations of HaloPROTAC3 may be necessary for successful and maximal degradation. Because HaloPROTAC3 binds irreversibly to HaloTag (Buckley et al., ent‐HaloPROTAC3 (Buckley et al., ent‐HaloPROTAC3 has significantly reduced affinity for VHL engagement (Buckley et al., With any PROTAC degradation study, it is important to be certain that observed target protein loss is due to the specific PROTAC mechanism. For endogenous HaloTag proteins, this can be achieved using For the alternate procedures utilizing HiBiT‐HaloTag fusion proteins, protein levels are easily measured with luminescence (Daniels et al., In the schematic shown in Figure ent‐HaloPROTAC3 at either 3 or 24 hr (Fig. As an example of HaloPROTAC3 degradation and phenotype studies of a target with no available degraders or specific PROTACs, a homozygous CRISPR clone of β‐catenin‐HaloTag‐HiBiT was generated in HEK293 cells stably expressing LgBiT. In Figure  hr Fig. , demonst hr Fig. . These r hr Fig.  and degr hr Fig. . Because hr Fig. , an orth hr Fig.  and E. U hr Fig. , includi hr Fig.  and E. T hr Fig.  and loss hr Fig. .ent‐HaloPROTAC3 (Fig. ent‐HaloPROTAC3 shows that protein loss of each of these targets is specific to HaloPROTAC3. Each of the HaloPROTAC3 degradation profiles was then used to calculate the degradation rate (Fig. 50, the concentration of HaloPROTAC3 that gave half the degradation maximum (Fig. 50 values for each of these targets indicates that it is primarily HaloTag which is driving the degradation, with minimal influence by the different targets. This is desirable for a fusion tag PROTAC system as it needs to be broadly applicable to numerous targets. The kinetic analysis also shows how the optimal dose of HaloPROTAC3 and time of treatment to achieve degradation can be clearly understood from the profiles, saving significant time and yielding more detailed information as compared to western blot analysis.To demonstrate the ability to degrade endogenous HaloTag proteins at a variety of cellular locations, as well as to understand the quantitative parameters of HaloPROTAC3 degradation, the HiBiT kinetic studies outlined in the optional live‐cell luminescence degradation detection of HiBiT‐HaloTag CRISPR insertion protocol steps were performed Fig. . For theAC3 Fig. ‐F to proAC3 Fig. ‐F. All tAC3 Fig. , includiAC3 Fig. . This isAC3 Fig. , suggestate Fig. , as wellmum Fig. , for allmum Fig. . The simF36V‐HiBiT at the C terminus in HEK293 cells (Fig. F36V has low efficiency of insertion and edited cells cannot easily be identified or fluorescently enriched from the CRISPR pool.To quantitatively compare HaloPROTAC3 degradation with the dTAG PROTAC system, CRISPR clones of the EPOP target protein were generated by insertion of either HaloTag‐HiBiT or FKBP12lls Fig. . These clls Fig.  or dTAG‐lls Fig.  were obtlls Fig. , and dTAlls Fig. , showed lls Fig. , which wlls Fig. . A hook lls Fig. . This rells Fig. . The Hallls Fig. . TogetheBasic Protocol Basic Protocol Optional HiBiT Lytic and kinetic protocols: The HiBiT lytic assay to determine insertion efficiency within CRISPR pools or measure degradation of HiBiT‐HaloTag target proteins takes 30‐60 min. The kinetic degradation assay takes 3 days to perform including plating, Endurazine equilibration, HaloPROTAC3 degradation, and data analysis.Elizabeth A. Caine: Data curation; formal analysis; investigation; writing‐original draft; writing‐review & editing. Sarah D. Mahan: Data curation; methodology. Rebecca L. Johnson: Methodology. Amanda N. Nieman: Methodology. Ngan Lam: Methodology; supervision. Curtis R. Warren: Conceptualization; supervision. Kristin M. Riching: Conceptualization; formal analysis; investigation. Marjeta Urh: Conceptualization; supervision; writing‐original draft; writing‐review & editing. Danette L. Daniels: Conceptualization; data curation; formal analysis; investigation; project administration.