Journal: bioRxiv

Article Title: A novel auxin-inducible degron system for rapid, cell cycle-specific targeted proteolysis

doi: 10.1101/2021.04.23.441203

Figure Lengend Snippet: Schematic representation of the design for Regulated OsTIR1 Levels of Expression based on the Cell Cycle Status (ROLECCS) variants. (A) OsTIR1-mEmerald protein (asynchronous ROLECCS, ROLECCS AS, 92 KDa) is stably expressed throughout the cell cycle. Upon auxin treatment, OsTIR1 enzymatic activity elicits the degradation of the mAID-tagged protein of interest (POI) in any cell, independently of the cell cycle status. (B) The expression of the ROLECCS G1 variant (OsTIR1-mEmerald-Cdt1, 103 KDa) is restricted to the G1/early S phase by the presence of the Cdt1 tag, when the SCF Skp E3 ligase activity is off. This, in turn, leads to auxin-dependent ubiquitylation and proteasome degradation of mAID-tagged POIs. In cells transitioning during S, G2 and M phases, SCF Skp activity is naturally restored, leading to ROLECCS G1 degradation by ubiquitylation, and stabilization of the POI even in the presence of auxin. (C) The Geminin tag of the ROLECCS G2 variant (OsTIR1-mEmerald-GEM, 105 KDa) ensures its restricted expression during the late S-G2-M phase, as APC Cdh -mediated ubiquitylation and degradation is rapidly triggered during M/G1 transition. Therefore, auxin treatment induces degradation of the POI exclusively in cells going through the late S-G2-M phase of the cell cycle.

Article Snippet: To construct pAAVS1-ROLECCS AS, pAAVS1-ROLECCS G1, and pAAVS1-ROLECCS G2 plasmids, multiple fragments were PCR amplified from different donor plasmids and assembled as follow: pMK232 CMV-OsTIR1-PURO (Addgene #72834 ) was used as donor plasmid for the expression of OsTIR1 from the AAVS1 locus, the mEmerald tag was PCR amplified from mEmerald-PLK1-N-16 vector (Addgene #54234; http://n2t.net/addgene:54234 ; RRID:Addgene_54234), while pEN435 - pCAGGS-TagBFP-hGeminin-2A-mCherry-hCdt1- rbgpA-Frt-PGK-EM7-PuroR-bpA-Frt Tigre targeting (Addgene #92139 ) was used as template for both hGeminin and hCdt1 tags.

Techniques: Expressing, Stable Transfection, Activity Assay, Variant Assay

Journal: bioRxiv

Article Title: A novel auxin-inducible degron system for rapid, cell cycle-specific targeted proteolysis

doi: 10.1101/2021.04.23.441203

Figure Lengend Snippet: The ROLECCS system performs a Boolean logic computation. The contemporary presence of auxin and appropriate phase of the cell cycle are both simultaneously required to lead to targeted protein degradation. ROLECCS G1 and G2 are stable only through specific phases of the cell cycle (G1/early S for ROLECCS G1, late S/G2/M for ROLECCS G2), therefore their biological activity is restricted to those phases. However, auxin is required to trigger OsTIR1- mediated protein ubiquitylation, allowing proteasomal degradation of the POI only “on demand”, and only in the appropriate phase of the cell cycle.

Article Snippet: To construct pAAVS1-ROLECCS AS, pAAVS1-ROLECCS G1, and pAAVS1-ROLECCS G2 plasmids, multiple fragments were PCR amplified from different donor plasmids and assembled as follow: pMK232 CMV-OsTIR1-PURO (Addgene #72834 ) was used as donor plasmid for the expression of OsTIR1 from the AAVS1 locus, the mEmerald tag was PCR amplified from mEmerald-PLK1-N-16 vector (Addgene #54234; http://n2t.net/addgene:54234 ; RRID:Addgene_54234), while pEN435 - pCAGGS-TagBFP-hGeminin-2A-mCherry-hCdt1- rbgpA-Frt-PGK-EM7-PuroR-bpA-Frt Tigre targeting (Addgene #92139 ) was used as template for both hGeminin and hCdt1 tags.

Techniques: Activity Assay

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Rapid COG Depletion in Mammalian Cell by Auxin-Inducible Degradation System

doi: 10.1007/978-1-0716-2639-9_23

Figure Lengend Snippet: Generation of hTERT RPE1 COG4 KO cell line expressing OsTIR1-9myc and COG4-mAID-mCherry: Cartoon image showing (A) The development of hTERT RPE1 cell line depleted for COG4 subunit by CRISPR-Cas9 approach and retroviral transduction resulting COG4 KO cells to express OsTIR1-9myc. (B) Creation of COG4-mAID stable cell line by rescuing the COG4 KO/OsTIR1-9myc cells by the lentiviral transduction to express COG4-mAID-mCherry under the control of COG4 promoter. (C) The experimental approach for COG4-mAID-mCherry rapid degradation by IAA (auxin) treatment

Article Snippet: Expression Plasmid 1 pBabe OsTIR1–9myc (PuroR) (Addgene #47328) [34].

Techniques: Expressing, CRISPR, Retroviral, Transduction, Stable Transfection, Control

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Rapid COG Depletion in Mammalian Cell by Auxin-Inducible Degradation System

doi: 10.1007/978-1-0716-2639-9_23

Figure Lengend Snippet: Acute depletion of COG4-mAID-mCherry displaces the B4GalT1 enzyme from the Golgi into CCD vesicles. (A) The hTERT RPE1 COG4 KO/OsTir1-9myc/hCOG4-mAid-mCherry cells were treated with 0.5 mM IAA for different times as indicated. Cells were collected and lysed, and the expression of B4GalT1 and actin was analyzed by WB. No depletion of B4GalT1 was observed within 2 h of treatment, but the B4GalT1 band was more diffuse at 24 h IAA treatment, indicating protein degradation. (B) IF analysis of untreated and IAA treated cells. The COG4-mAID clone was treated 0.5 mM IAA for 1 h. The cells were fixed and stained for GM130 (green) and B4GalT1 (as red). Note that the acute depletion of COG4-mAID-mCherry displaced the enzyme from the Golgi into vesicle-like structures. Scale bars, 20 μm. (C) Quantification of colocalization between GM130 and B4GalT1 from n = 2 independent experiment with >30 cells analyzed. ****, P < 0.0001, significant. Error bar represents mean ± SD. The green and red channels are presented in inverted black and white mode, whereas the merged view is shown in RGB mode. Scale bars, 20 μm

Article Snippet: Expression Plasmid 1 pBabe OsTIR1–9myc (PuroR) (Addgene #47328) [34].

Techniques: Expressing, Staining

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Rapid COG Depletion in Mammalian Cell by Auxin-Inducible Degradation System

doi: 10.1007/978-1-0716-2639-9_23

Figure Lengend Snippet: Auxin (IAA) treatment induces rapid depletion of hCOG4-mAID-mCherry. (A) The hTERT RPE1 COG4 KO/OsTir1-9myc/hCOG4-mAid-mCherry cells were treated with 0.5 mM IAA for different times as indicated. Cells were collected and lysed, and the expression of wild-type and hybrid COG4 protein was analyzed by WB using an anti-COG4 antibody. (B) Quantification of the COG4 protein level at different time points after IAA treatment. Three independent experiments have been performed. (C) IF analysis of COG4-mAID-mCherry depletion after IAA treatment. The COG4 mAID clone was treated with IAA for 1 h. Untreated cells were used as a control. The cells were fixed and stained for GM130 (green). Scale bars, 20 μm

Article Snippet: Expression Plasmid 1 pBabe OsTIR1–9myc (PuroR) (Addgene #47328) [34].

Techniques: Expressing, Control, Staining

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Rapid degradation of EGFP fused to a mAID degron. A) A vector for expressing both OsTIR(F74G) and EGFP fused to a mAID degron and a nuclear export sequence (NES), separated by a P2A sequence. Same construct as described by , now in a lentiviral expression vector. B) Examples of neurons expressing OsTIR1(F74G) and EGFP:mAID (yellow) either exposed (right hand panels) or not exposed (left panels) to 200nM 5-Ph-IAA. Magenta dots are fluorescent background objects, used here to show that the loss of EGFP fluorescence is not a result of focal drift. Bar, 20µm. C) EGFP fluorescence measured at the cell bodies of 17 and 13 neurons exposed or not exposed to 5-Ph-IAA, respectively (thin gray lines). Fluorescence values for each neuron normalized to fluorescence measured at the last time point before 5-Ph-IAA was added. Thick red (5-Ph-IAA treated) and blue (untreated) lines represent population averages for each condition. Data from two separate experiments.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Plasmid Preparation, Expressing, Sequencing, Construct, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Rapid degradation of postsynaptic scaffold proteins fused to mTurquoise2 and a mAID degron. A) Illustration of the PSD:95, mTurquoise2 and mAID fusion protein (PSD-95:mTurq2:mAID). B) A rat cortical neuron in culture expressing PSD-95:mTurq2:mAID as well as OsTIR1-P2A-EGFP:mAID (see ; EGFP fluorescence not shown). C) Region in yellow rectangle in B at greater detail, before, after addition of 5-Ph-IAA, and after washing out the 5-Ph-IAA. Note the nearly complete loss of PSD-95:mTurq2:mAID fluorescence, and its recovery after several days. D) PSD-95:mTurq2:mAID fluorescence measured at 30 synapses of each neuron tracked throughout the experiments. Each thin gray line is the average fluorescence measured for the synapses of one neuron. Fluorescence values for each neuron were normalized to the fluorescence measured at the last time point before 5-Ph-IAA was added. Thick red line - population average (19 neurons from 3 independent experiments, 570 synapses in total). E-H) As in A-D for mAID:mTurq2:GKAP. 14 neurons from 2 independent experiments, 10-19 synapses per neuron, 199 in total). I-L) As in A-D for mAID:mTurq2:Gephyrin. 24 neurons from 3 independent experiments, 30 synapses per neuron, 720 synapses in total. Scale bars: 20 µm (B,F,J) and 10 µm C,G,K.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Rapid degradation of postsynaptic scaffold proteins fused to HaloTag protein and a mAID degron. A) Illustration of the PSD-95 - HaloTag - mAID fusion protein (PSD-95:HT:mAID). B) A rat cortical neuron in culture expressing PSD-95:HT:mAID as well as OsTIR1-P2A-EGFP:mAID (EGFP fluorescence not shown). PSD-95:HT:mAID was rendered visible by labeling with JF635-HT (100nM). C) Region in yellow rectangle in B at greater detail, before, after addition of 5-Ph-IAA, and after washing out 5-Ph-IAA. Note the nearly complete loss of JF635-HT fluorescence, and its recovery after several days (following a second labeling with JF635-HT). D) JF635-HT fluorescence measured at 11-30 synapses of each neuron (443 in total) tracked throughout the experiments. Each thin gray line is the average JF635-HT fluorescence measured for the synapses of one neuron. Fluorescence values for each neuron were normalized to the fluorescence measured at the last time point before 5-Ph-IAA was added. Thick red line is the population average (24 neurons from 3 independent experiments). E-H) As in A-D for GephyrinA29:mAID:HT. 18 neurons from 3 independent experiments. Scale bars: 20µm (B,F) and 10µm C,G.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Fluorescence, Labeling

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: PSD-95:mTurq2:mAID loss rate dependencies. A) Rates of synaptic PSD-95:mTurq2:mAID loss (in fluorescence units / h) were calculated for each neuron coexpressing PSD-95:mTurq2:mAID and OsTIR1-P2A-mCherry by a fitting a line to mTurq2 fluorescence measurements made during the first 5 time points following exposure to 5-Ph-IAA (Inset). Loss rates were then plotted against initial PSD-95:mTurq2:mAID levels in the same neurons. The correlation between these two measures was -0.747 (34 neurons from 5 experiments). B) Similar plots comparing mTurq2 loss rate to mCherry fluorescence measured for the same neuron, with mCherry fluorescence serving as a surrogate for OsTIR1 expression. The correlation between these two measures was -0.651 (27 neurons from 4 experiments).

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Fluorescence, Expressing

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: AID2-mediated elimination and recovery of a soluble protein in vivo . A ) Flow of experiment. C57BL/6J mice (n = 3) were injected with AAV vectors to cytosolically express mAID:GFP, OsTir1 and a stable nuclear reference marker (H2B:mCherry) in the auditory cortex. Animals underwent cranial window implantation and longitudinal two-photon imaging after sham (PBS) or 5-Ph-IAA treatment. B ) Exemplary fields of view, showing stable mAID:EGFP fluorescence after sham treatment and vastly depleted mAID:EGFP fluorescence after auxin treatment. C ) Quantification of the normalized cytosolic mAID:EGFP fluorescence (see Methods) over the course of imaging (n=91 sham neurons vs. 111 5-Ph-IAA treated neurons), showing a swift depletion over the course of few hours and a subsequent recovery after approximately three days (***: p<0.001, Wilcoxon rank sum test of sham vs. 5-Ph-IAA).

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: In Vivo, Injection, Marker, Imaging, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Acute elimination of PSD-95:mTurq2:mAID is followed by loss of AMPARs at the same synapses. A) Top panels: A rat cortical neuron in culture co-expressing PSD-95:mTurq2:mAID, SEpH:GluA2 and OsTIR1-P2A-mCherry (not shown). Bottom panels: Region in yellow rectangle at greater detail, before, and after addition of 5-Ph-IAA. Note that the near complete loss of PSD-95:mTurq2:mAID is associated with a modest reduction in SEpH:GluA2 fluorescence. Scale bars: 10 µm. B) PSD-95:mTurq2:mAID fluorescence measured at 16-41 synapses of each neuron (455 in total) tracked throughout the experiments. Each thin gray line is the average fluorescence measured for the synapses of one neuron (18 neurons from 3 experiments). Fluorescence was normalized to fluorescence measured at time point just before 5-Ph-IAA addition. Thick magenta line is the population average. A subset of neurons was imaged only once every 12 (instead of 3) h to minimize potential confounds related to photobleaching (open diamonds; 10 neurons from the same 3 experiments). C) changes in SEpH:GluA2 fluorescence at the same synapses and neurons of B. Thick brown line is the population average. D) SEpH:GluA2 fluorescence measured at 14-30 synapses of neurons positive for SEpH:GluA2 and OsTIR1-P2A-mCherry but negative for PSD-95:mTurq2:mAID (222 in total). Each thin gray line is the average fluorescence measured for the synapses of one neuron (10 neurons from 3 experiments). Thick gray line is the population average Open diamonds represent measurements made in a subset of cells imaged only once every 12 h (5 neurons from the same experiments). E) Pooled data. All error bars are standard deviations, not SEM. Test for difference between PSD-95:mTurq2:mAID positive and negative cells – unpaired t-test, without assuming equal variances; applied to data obtained at last time point.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Quantification of PSD-95 overexpression. Networks of neurons expressing PSD-95:mTurq2:mAID, SEpH:GluA2 and OsTIR1-P2A-mCherry as well as age matched naïve networks from the same cell culture preparations were fixed and stained against PSD-95 (FluoTag®-X2 anti-PSD95 Alexa 647; NanoTag Biotechnologies #N3702-AF647-L; 1:200). A) Naïve (uninfected neurons) fixed and immunolabeled against PSD-95. B) A neuron expressing PSD-95:mTurq2:mAID fixed and immunolabeled against PSD-95. C) PSD-95:mTurq2:mAID fluorescence of the same synapses as in B. Note that in this particular field of view, all synapses were PSD-95:mTurq2:mAID positive. D) Comparison of anti-PSD-95 immunofluorescence of postsynaptic sites positive for PSD95:mTurq:mAID in the triple infected preparations to that of postsynaptic densities in the naïve ones (15,839 and 53,913 synapses from 24 and 31 fields of view, respectively; two separate experiments, shown separately). Every data point is the average fluorescence (arbitrary units) for one field of view. Columns and error bars show means and standard deviations. Correlations (Pearson’s) between PSD-95:mTurq2:mAID and anti-PSD-95 fluorescence on a synapse by synapses basis were 0.86 and 0.80 for the two experiments, indicating that anti-PSD-95 fluorescence provided a good readout of PSD-95 expression levels.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Over Expression, Expressing, Cell Culture, Staining, Immunolabeling, Fluorescence, Comparison, Immunofluorescence, Infection

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Acute elimination of GephyrinA29:mAID:HT is followed by loss of GABARs at the same synapses. A) Top panels: A rat cortical neuron in culture co-expressing GephyrinA29:mAID:HT (labeled with JF635-HT), SEpH:GABA A Rα 2 and OsTIR1-P2A-mCherry (not shown). Bottom panels: Region in yellow rectangle at greater detail, before, and after addition of 5-Ph-IAA. Note that the near complete loss of JF635-HT fluorescence (presumably reflecting GephyrinA29:mAID:HT degradation) is associated with a modest reduction in SEpH:GABA A Rα 2 fluorescence. Scale bars: 10 µm (top panels) 5µm (bottom panels). B) JF635-HT fluorescence measured at 50 synapses of each neuron tracked throughout the experiments. Each thin gray line is the average JF635-HT fluorescence measured for the synapses of one neuron (18 neurons from 3 experiments). Thick magenta line is the population average . C) changes in SEpH:GABA A Rα 2 fluorescence at the same synapses and neurons of B. Thick brown line is the population average. D) SEpH:GABA A Rα 2 fluorescence measured at 150 synapses of neurons positive for SEpH:GABA A Rα 2 and OsTIR1-P2A-mCherry but negative for GephyrinA29:mAID:HT. Each thin gray line is the average fluorescence measured for the synapses of one neuron (3 neurons from 2 experiments). Thick gray line is the population average. E) Pooled data. Error bars are standard deviations. Test for difference between GephyrinA29:mAID:HT positive and negative cells – unpaired t-test, without assuming equal variances; applied to data of last time point.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Labeling, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Quantification of Gephyrin overexpression. Preparations containing neurons expressing GephyrinA29:mAID:HT, SEpH:GABA A Rα 2 and OsTIR1-P2A-mCherry as well as age matched naïve networks from the same cell culture preparations were fixed and stained against Gephyrin (monoclonal mouse anti gephyrin Synaptic Systems #147111). Infected networks were labeled before fixation with JF552-HT. A) Naïve (uninfected neurons) fixed and immunolabeled against gephyrin. B) A neuron expressing GephyrinA29:mAID:HT fixed and immunolabeled against gephyrin. C) GephyrinA29:mAID:HT + JF552-HT fluorescence of the same synapses as in B. D) Comparison of Anti-Gephyrin immunofluorescence of postsynaptic sites positive to JF552-HT fluorescence in the triple infected networks to that of postsynaptic densities in the naïve networks (6,204 and 4,626 synapses from 16 and 16 fields of view, respectively; two separate experiments, shown separately). Every data point is the average fluorescence (arbitrary units) for one field of view. Columns and error bars show means and standard deviations. Correlation (Pearson’s) between JF552-HT fluorescence and anti-Gephyrin fluorescence on a synapse by synapses basis was 0.50, indicating that anti-Gephyrin fluorescence provided a reasonable, if imperfect, readout of gephyrin expression levels, possibly affected by the use of a HT - HT ligand pair rather than a fluorescent protein

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Over Expression, Expressing, Cell Culture, Staining, Infection, Labeling, Immunolabeling, Fluorescence, Comparison, Immunofluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Acute elimination of mAID:mTurq2:Gephyrin is followed by loss of GABA receptors at the same synapses. A) Top panels: A rat cortical neuron in culture co-expressing mAID:mTurq2:Gephyrin, SEpH:GABA A Rα 2 and OsTIR1-P2A-mCherry (not shown). Bottom panels: Region in yellow rectangle at greater detail, before, and after addition of 5-Ph-IAA. Note that the near complete loss of fluorescence (presumably reflecting mAID:mTurq2:Gephyrin degradation) is associated with a partial reduction in SEpH:GABA A Rα 2 fluorescence. Scale bars: 10µm (top panels) 5µm (bottom panels). B) mAID:mTurq2:Gephyrin fluorescence measured at 50 synapses of each neuron tracked throughout the experiments. Each thin gray line is the average mAID:mTurq2:Gephyrin fluorescence (normalized to the time point just before 5-Ph-IAA addition) measured for the synapses of one neuron (18 neurons from 3 experiments). Thick magenta line is the population average . C) changes in SEpH:GABA A Rα 2 fluorescence at the same synapses and neurons of B. Thick brown line is the population average. D) SEpH:GABA A Rα 2 fluorescence measured at 200 synapses of neurons positive for SEpH:GABA A Rα 2 and OsTIR1-P2A-mCherry but negative for mAID:mTurq2:Gephyrin. Each thin gray line is the average fluorescence measured for the synapses of one neuron (4 neurons from 1 experiment). Thick gray line is the population average. E) Pooled data. Error bars are standard deviations. Test for difference between mAID:mTurq2:Gephyrin positive and negative cells – unpaired t-test, without assuming equal variances; applied to data from last time point.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Acute elimination of PSD-95:mTurq2:mAID is followed by ‘substitution’ with GKAP. A) A rat cortical neuron in culture co-expressing PSD-95:mTurq2:mAID (left) and mCit:GKAP (middle) as well as OsTIR1-P2A-mCherry (not shown). B) 5-Ph-IAA induced nearly complete loss of PSD-95:mTurq2:mAID, and increased synaptic levels of mCit:GKAP at the same synapses. Scale bar: 20 µm. C) Changes in mCit:GKAP fluorescence measured at 13-40 synapses of each neuron tracked throughout the experiments (303 in total). Each thin gray line is the average normalized fluorescence measured for the synapses of one neuron (12 neurons from 2 experiments). Thick brown line is the population average. D) PSD-95:mTurq2:mAID fluorescence measured at the same synapses and neurons of C. Thick magenta line is the population average. E) mCit:GKAP fluorescence measured at 18-33 synapses of neurons positive for mCit:GKAP and OsTIR1-P2A-mCherry but negative for PSD-95:mTurq2:mAID (147 in total). Each thin gray line is the average fluorescence measured for the synapses of one neuron (6 neurons from 2 experiments). Thick gray line is the population average. F) Pooled data. Error bars are standard deviations. Test for difference between PSD-95:mTurq2:mAID positive and negative cells – unpaired t-test, without assuming equal variances; applied to data of last time point. See also .

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Acute elimination of PSD-95:mTurq2:mAID is followed by ‘substitution’ with GKAP. A) Cortical neurons in culture were infected with lentiviral expression vectors encoding for PSD-95:mTurq2:mAID, mCit:GKAP and OsTIR1-P2A-mCherry. Changes in mCit:GKAP fluorescence were measured at synapses in fields of view containing neurons expressing all three exogenous proteins. Here, unlike , synapses were not tracked individually but located programmatically anew at each time step, resulting in an unbiased selection of synapses but with no distinction between synapses belonging to triple expressing neurons and synapses belonging to other neurons in the same fields of view. Each thin gray line is the average fluorescence measured for the synapses in one field of view (13 fields of view from 3 experiments). Thick brown line is the population average. B) mCit:GKAP fluorescence measured at synapses in fields of view containing neurons positive for mCit:GKAP and OsTIR1-P2A-mCherry but negative for PSD-95:mTurq2:mAID. Each thin gray line is the average fluorescence measured for the synapses of one neuron (9 neurons from 3 experiments). Thick gray line is the population average. C) PSD-95:mTurq2:mAID fluorescence measured at the same synapses and neurons of A. Thick magenta line is the population average. D) Pooled data. Error bars are standard deviations. Test for difference between PSD-95:mTurq2:mAID positive and negative cells – unpaired t-test, without assuming equal variances; applied to data from t=30 h.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Infection, Expressing, Fluorescence, Selection

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Acute elimination of PSD-95:mTurq2:mAID is followed by rapid replacement with PSD-95:mCitrine. A) A rat cortical neuron in culture co-expressing PSD-95:mTurq2:mAID (left) and PSD-95:mCitrine (middle) as well as OsTIR1-P2A-mCherry (not shown). B) 5-Ph-IAA induced nearly complete loss of PSD-95:mTurq2:mAID, and increased synaptic levels of PSD-95:mCitrine at the same synapses. Scale bar: 20 µm. C) Changes in PSD-95:mCitrine fluorescence measured at 15-34 synapses of each neuron tracked throughout the experiments (485 in total). Each thin gray line is the average normalized fluorescence measured for the synapses of one neuron (22 neurons from 3 experiments). Thick brown line is the population average. D) PSD-95:mTurq2:mAID fluorescence measured at the same synapses and neurons of C. Thick magenta line is the population average. E) PSD-95:mCitrine fluorescence measured at 14-42 synapses of neurons positive for PSD-95:mCitrine and OsTIR1-P2A-mCherry but negative for PSD-95:mTurq2:mAID (380 in total). Each thin gray line is the average fluorescence measured for the synapses of one neuron (14 neurons from 3 experiments). Thick gray line is the population average. F) Pooled data. Error bars are standard deviations. Test for difference between PSD-95:mTurq2:mAID positive and negative cells – unpaired t-test, without assuming equal variances; applied to data from last time point.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Acute elimination of mAID:mTurq2:GKAP is followed by concomitant loss of synaptic PSD-95 and elevated levels of cytosolic PSD-95. A) A rat cortical neuron in culture co-expressing PSD-95:mCit (left) mAID:mTurq2:GKAP (middle) as well as OsTIR1-P2A-mCherry (not shown). B) 5-Ph-IAA induced nearly complete loss of mAID:mTurq2:GKAP, loss of PSD-95:mCit from the same synapses and elevated levels of cytosolic PSD-95:mCit. Scale bar: 20 µm. C) Changes in mAID:mTurq2:GKAP fluorescence measured at 24-78 synapses of each neuron tracked throughout the experiments (1,723 synapses in total). Each thin gray line is the average normalized fluorescence measured for the synapses of one neuron (40 neurons from 5 experiments). Thick brown line is the population average. D) PSD-95:mCit fluorescence measured at the same synapses and neurons of C. Thick magenta line is the population average. E) PSD-95:mCit fluorescence measured at 21-64 synapses of neurons positive for PSD-95:mCit and OsTIR1-P2A-mCherry but negative for mAID:mTurq2:GKAP (752 synapses in total). Each thin gray line is the average fluorescence measured for the synapses of one neuron (18 neurons from 5 experiments). Thick gray line is the population average. F) Changes in cytosolic levels of PSD-95:mCit measured at the cell soma (31 neurons from 5 experiments). Dashed thick magenta line is the population average. G) Changes in cytosolic PSD-95:mCit measured in the cell bodies of neurons positive for PSD-95:mCit and OsTIR1-P2A-mCherry but negative for mAID:mTurq2:GKAP (14 neurons from 5 experiments). Dashed thick gray line is the population average. H) Pooled data. Dashed lines with filled triangles represent cytosolic PSD-95:mCit levels. Error bars are standard deviations. Tests for difference between PSD-95:mTurq2:mAID positive and negative cells – unpaired t-tests, without assuming equal variances; applied to data of last time points.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Rapid degradation of EGFP fused to a mAID degron. A) A vector for expressing both OsTIR(F74G) and EGFP fused to a mAID degron and a nuclear export sequence (NES), separated by a P2A sequence. Same construct as described by , now in a lentiviral expression vector. B) Examples of neurons expressing OsTIR1(F74G) and EGFP:mAID (yellow) either exposed (right hand panels) or not exposed (left panels) to 200nM 5-Ph-IAA. Magenta dots are fluorescent background objects, used here to show that the loss of EGFP fluorescence is not a result of focal drift. Bar, 20µm. C) EGFP fluorescence measured at the cell bodies of 17 and 13 neurons exposed or not exposed to 5-Ph-IAA, respectively (thin gray lines). Fluorescence values for each neuron normalized to fluorescence measured at the last time point before 5-Ph-IAA was added. Thick red (5-Ph-IAA treated) and blue (untreated) lines represent population averages for each condition. Data from two separate experiments.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Plasmid Preparation, Expressing, Sequencing, Construct, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Rapid degradation of postsynaptic scaffold proteins fused to mTurquoise2 and a mAID degron. A) Illustration of the PSD:95, mTurquoise2 and mAID fusion protein (PSD-95:mTurq2:mAID). B) A rat cortical neuron in culture expressing PSD-95:mTurq2:mAID as well as OsTIR1-P2A-EGFP:mAID (see ; EGFP fluorescence not shown). C) Region in yellow rectangle in B at greater detail, before, after addition of 5-Ph-IAA, and after washing out the 5-Ph-IAA. Note the nearly complete loss of PSD-95:mTurq2:mAID fluorescence, and its recovery after several days. D) PSD-95:mTurq2:mAID fluorescence measured at 30 synapses of each neuron tracked throughout the experiments. Each thin gray line is the average fluorescence measured for the synapses of one neuron. Fluorescence values for each neuron were normalized to the fluorescence measured at the last time point before 5-Ph-IAA was added. Thick red line - population average (19 neurons from 3 independent experiments, 570 synapses in total). E-H) As in A-D for mAID:mTurq2:GKAP. 14 neurons from 2 independent experiments, 10-19 synapses per neuron, 199 in total). I-L) As in A-D for mAID:mTurq2:Gephyrin. 24 neurons from 3 independent experiments, 30 synapses per neuron, 720 synapses in total. Scale bars: 20 µm (B,F,J) and 10 µm C,G,K.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Rapid degradation of postsynaptic scaffold proteins fused to HaloTag protein and a mAID degron. A) Illustration of the PSD-95 - HaloTag - mAID fusion protein (PSD-95:HT:mAID). B) A rat cortical neuron in culture expressing PSD-95:HT:mAID as well as OsTIR1-P2A-EGFP:mAID (EGFP fluorescence not shown). PSD-95:HT:mAID was rendered visible by labeling with JF635-HT (100nM). C) Region in yellow rectangle in B at greater detail, before, after addition of 5-Ph-IAA, and after washing out 5-Ph-IAA. Note the nearly complete loss of JF635-HT fluorescence, and its recovery after several days (following a second labeling with JF635-HT). D) JF635-HT fluorescence measured at 11-30 synapses of each neuron (443 in total) tracked throughout the experiments. Each thin gray line is the average JF635-HT fluorescence measured for the synapses of one neuron. Fluorescence values for each neuron were normalized to the fluorescence measured at the last time point before 5-Ph-IAA was added. Thick red line is the population average (24 neurons from 3 independent experiments). E-H) As in A-D for GephyrinA29:mAID:HT. 18 neurons from 3 independent experiments. Scale bars: 20µm (B,F) and 10µm C,G.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Fluorescence, Labeling

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: PSD-95:mTurq2:mAID loss rate dependencies. A) Rates of synaptic PSD-95:mTurq2:mAID loss (in fluorescence units / h) were calculated for each neuron coexpressing PSD-95:mTurq2:mAID and OsTIR1-P2A-mCherry by a fitting a line to mTurq2 fluorescence measurements made during the first 5 time points following exposure to 5-Ph-IAA (Inset). Loss rates were then plotted against initial PSD-95:mTurq2:mAID levels in the same neurons. The correlation between these two measures was -0.747 (34 neurons from 5 experiments). B) Similar plots comparing mTurq2 loss rate to mCherry fluorescence measured for the same neuron, with mCherry fluorescence serving as a surrogate for OsTIR1 expression. The correlation between these two measures was -0.651 (27 neurons from 4 experiments).

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Fluorescence, Expressing

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: AID2-mediated elimination and recovery of a soluble protein in vivo . A ) Flow of experiment. C57BL/6J mice (n = 3) were injected with AAV vectors to cytosolically express mAID:GFP, OsTir1 and a stable nuclear reference marker (H2B:mCherry) in the auditory cortex. Animals underwent cranial window implantation and longitudinal two-photon imaging after sham (PBS) or 5-Ph-IAA treatment. B ) Exemplary fields of view, showing stable mAID:EGFP fluorescence after sham treatment and vastly depleted mAID:EGFP fluorescence after auxin treatment. C ) Quantification of the normalized cytosolic mAID:EGFP fluorescence (see Methods) over the course of imaging (n=91 sham neurons vs. 111 5-Ph-IAA treated neurons), showing a swift depletion over the course of few hours and a subsequent recovery after approximately three days (***: p<0.001, Wilcoxon rank sum test of sham vs. 5-Ph-IAA).

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: In Vivo, Injection, Marker, Imaging, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Acute elimination of PSD-95:mTurq2:mAID is followed by loss of AMPARs at the same synapses. A) Top panels: A rat cortical neuron in culture co-expressing PSD-95:mTurq2:mAID, SEpH:GluA2 and OsTIR1-P2A-mCherry (not shown). Bottom panels: Region in yellow rectangle at greater detail, before, and after addition of 5-Ph-IAA. Note that the near complete loss of PSD-95:mTurq2:mAID is associated with a modest reduction in SEpH:GluA2 fluorescence. Scale bars: 10 µm. B) PSD-95:mTurq2:mAID fluorescence measured at 16-41 synapses of each neuron (455 in total) tracked throughout the experiments. Each thin gray line is the average fluorescence measured for the synapses of one neuron (18 neurons from 3 experiments). Fluorescence was normalized to fluorescence measured at time point just before 5-Ph-IAA addition. Thick magenta line is the population average. A subset of neurons was imaged only once every 12 (instead of 3) h to minimize potential confounds related to photobleaching (open diamonds; 10 neurons from the same 3 experiments). C) changes in SEpH:GluA2 fluorescence at the same synapses and neurons of B. Thick brown line is the population average. D) SEpH:GluA2 fluorescence measured at 14-30 synapses of neurons positive for SEpH:GluA2 and OsTIR1-P2A-mCherry but negative for PSD-95:mTurq2:mAID (222 in total). Each thin gray line is the average fluorescence measured for the synapses of one neuron (10 neurons from 3 experiments). Thick gray line is the population average Open diamonds represent measurements made in a subset of cells imaged only once every 12 h (5 neurons from the same experiments). E) Pooled data. All error bars are standard deviations, not SEM. Test for difference between PSD-95:mTurq2:mAID positive and negative cells – unpaired t-test, without assuming equal variances; applied to data obtained at last time point.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Quantification of PSD-95 overexpression. Networks of neurons expressing PSD-95:mTurq2:mAID, SEpH:GluA2 and OsTIR1-P2A-mCherry as well as age matched naïve networks from the same cell culture preparations were fixed and stained against PSD-95 (FluoTag®-X2 anti-PSD95 Alexa 647; NanoTag Biotechnologies #N3702-AF647-L; 1:200). A) Naïve (uninfected neurons) fixed and immunolabeled against PSD-95. B) A neuron expressing PSD-95:mTurq2:mAID fixed and immunolabeled against PSD-95. C) PSD-95:mTurq2:mAID fluorescence of the same synapses as in B. Note that in this particular field of view, all synapses were PSD-95:mTurq2:mAID positive. D) Comparison of anti-PSD-95 immunofluorescence of postsynaptic sites positive for PSD95:mTurq:mAID in the triple infected preparations to that of postsynaptic densities in the naïve ones (15,839 and 53,913 synapses from 24 and 31 fields of view, respectively; two separate experiments, shown separately). Every data point is the average fluorescence (arbitrary units) for one field of view. Columns and error bars show means and standard deviations. Correlations (Pearson’s) between PSD-95:mTurq2:mAID and anti-PSD-95 fluorescence on a synapse by synapses basis were 0.86 and 0.80 for the two experiments, indicating that anti-PSD-95 fluorescence provided a good readout of PSD-95 expression levels.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Over Expression, Expressing, Cell Culture, Staining, Immunolabeling, Fluorescence, Comparison, Immunofluorescence, Infection

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Acute elimination of GephyrinA29:mAID:HT is followed by loss of GABARs at the same synapses. A) Top panels: A rat cortical neuron in culture co-expressing GephyrinA29:mAID:HT (labeled with JF635-HT), SEpH:GABA A Rα 2 and OsTIR1-P2A-mCherry (not shown). Bottom panels: Region in yellow rectangle at greater detail, before, and after addition of 5-Ph-IAA. Note that the near complete loss of JF635-HT fluorescence (presumably reflecting GephyrinA29:mAID:HT degradation) is associated with a modest reduction in SEpH:GABA A Rα 2 fluorescence. Scale bars: 10 µm (top panels) 5µm (bottom panels). B) JF635-HT fluorescence measured at 50 synapses of each neuron tracked throughout the experiments. Each thin gray line is the average JF635-HT fluorescence measured for the synapses of one neuron (18 neurons from 3 experiments). Thick magenta line is the population average . C) changes in SEpH:GABA A Rα 2 fluorescence at the same synapses and neurons of B. Thick brown line is the population average. D) SEpH:GABA A Rα 2 fluorescence measured at 150 synapses of neurons positive for SEpH:GABA A Rα 2 and OsTIR1-P2A-mCherry but negative for GephyrinA29:mAID:HT. Each thin gray line is the average fluorescence measured for the synapses of one neuron (3 neurons from 2 experiments). Thick gray line is the population average. E) Pooled data. Error bars are standard deviations. Test for difference between GephyrinA29:mAID:HT positive and negative cells – unpaired t-test, without assuming equal variances; applied to data of last time point.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Labeling, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Quantification of Gephyrin overexpression. Preparations containing neurons expressing GephyrinA29:mAID:HT, SEpH:GABA A Rα 2 and OsTIR1-P2A-mCherry as well as age matched naïve networks from the same cell culture preparations were fixed and stained against Gephyrin (monoclonal mouse anti gephyrin Synaptic Systems #147111). Infected networks were labeled before fixation with JF552-HT. A) Naïve (uninfected neurons) fixed and immunolabeled against gephyrin. B) A neuron expressing GephyrinA29:mAID:HT fixed and immunolabeled against gephyrin. C) GephyrinA29:mAID:HT + JF552-HT fluorescence of the same synapses as in B. D) Comparison of Anti-Gephyrin immunofluorescence of postsynaptic sites positive to JF552-HT fluorescence in the triple infected networks to that of postsynaptic densities in the naïve networks (6,204 and 4,626 synapses from 16 and 16 fields of view, respectively; two separate experiments, shown separately). Every data point is the average fluorescence (arbitrary units) for one field of view. Columns and error bars show means and standard deviations. Correlation (Pearson’s) between JF552-HT fluorescence and anti-Gephyrin fluorescence on a synapse by synapses basis was 0.50, indicating that anti-Gephyrin fluorescence provided a reasonable, if imperfect, readout of gephyrin expression levels, possibly affected by the use of a HT - HT ligand pair rather than a fluorescent protein

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Over Expression, Expressing, Cell Culture, Staining, Infection, Labeling, Immunolabeling, Fluorescence, Comparison, Immunofluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Acute elimination of mAID:mTurq2:Gephyrin is followed by loss of GABA receptors at the same synapses. A) Top panels: A rat cortical neuron in culture co-expressing mAID:mTurq2:Gephyrin, SEpH:GABA A Rα 2 and OsTIR1-P2A-mCherry (not shown). Bottom panels: Region in yellow rectangle at greater detail, before, and after addition of 5-Ph-IAA. Note that the near complete loss of fluorescence (presumably reflecting mAID:mTurq2:Gephyrin degradation) is associated with a partial reduction in SEpH:GABA A Rα 2 fluorescence. Scale bars: 10µm (top panels) 5µm (bottom panels). B) mAID:mTurq2:Gephyrin fluorescence measured at 50 synapses of each neuron tracked throughout the experiments. Each thin gray line is the average mAID:mTurq2:Gephyrin fluorescence (normalized to the time point just before 5-Ph-IAA addition) measured for the synapses of one neuron (18 neurons from 3 experiments). Thick magenta line is the population average . C) changes in SEpH:GABA A Rα 2 fluorescence at the same synapses and neurons of B. Thick brown line is the population average. D) SEpH:GABA A Rα 2 fluorescence measured at 200 synapses of neurons positive for SEpH:GABA A Rα 2 and OsTIR1-P2A-mCherry but negative for mAID:mTurq2:Gephyrin. Each thin gray line is the average fluorescence measured for the synapses of one neuron (4 neurons from 1 experiment). Thick gray line is the population average. E) Pooled data. Error bars are standard deviations. Test for difference between mAID:mTurq2:Gephyrin positive and negative cells – unpaired t-test, without assuming equal variances; applied to data from last time point.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Acute elimination of PSD-95:mTurq2:mAID is followed by ‘substitution’ with GKAP. A) A rat cortical neuron in culture co-expressing PSD-95:mTurq2:mAID (left) and mCit:GKAP (middle) as well as OsTIR1-P2A-mCherry (not shown). B) 5-Ph-IAA induced nearly complete loss of PSD-95:mTurq2:mAID, and increased synaptic levels of mCit:GKAP at the same synapses. Scale bar: 20 µm. C) Changes in mCit:GKAP fluorescence measured at 13-40 synapses of each neuron tracked throughout the experiments (303 in total). Each thin gray line is the average normalized fluorescence measured for the synapses of one neuron (12 neurons from 2 experiments). Thick brown line is the population average. D) PSD-95:mTurq2:mAID fluorescence measured at the same synapses and neurons of C. Thick magenta line is the population average. E) mCit:GKAP fluorescence measured at 18-33 synapses of neurons positive for mCit:GKAP and OsTIR1-P2A-mCherry but negative for PSD-95:mTurq2:mAID (147 in total). Each thin gray line is the average fluorescence measured for the synapses of one neuron (6 neurons from 2 experiments). Thick gray line is the population average. F) Pooled data. Error bars are standard deviations. Test for difference between PSD-95:mTurq2:mAID positive and negative cells – unpaired t-test, without assuming equal variances; applied to data of last time point. See also .

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Acute elimination of PSD-95:mTurq2:mAID is followed by ‘substitution’ with GKAP. A) Cortical neurons in culture were infected with lentiviral expression vectors encoding for PSD-95:mTurq2:mAID, mCit:GKAP and OsTIR1-P2A-mCherry. Changes in mCit:GKAP fluorescence were measured at synapses in fields of view containing neurons expressing all three exogenous proteins. Here, unlike , synapses were not tracked individually but located programmatically anew at each time step, resulting in an unbiased selection of synapses but with no distinction between synapses belonging to triple expressing neurons and synapses belonging to other neurons in the same fields of view. Each thin gray line is the average fluorescence measured for the synapses in one field of view (13 fields of view from 3 experiments). Thick brown line is the population average. B) mCit:GKAP fluorescence measured at synapses in fields of view containing neurons positive for mCit:GKAP and OsTIR1-P2A-mCherry but negative for PSD-95:mTurq2:mAID. Each thin gray line is the average fluorescence measured for the synapses of one neuron (9 neurons from 3 experiments). Thick gray line is the population average. C) PSD-95:mTurq2:mAID fluorescence measured at the same synapses and neurons of A. Thick magenta line is the population average. D) Pooled data. Error bars are standard deviations. Test for difference between PSD-95:mTurq2:mAID positive and negative cells – unpaired t-test, without assuming equal variances; applied to data from t=30 h.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Infection, Expressing, Fluorescence, Selection

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Acute elimination of PSD-95:mTurq2:mAID is followed by rapid replacement with PSD-95:mCitrine. A) A rat cortical neuron in culture co-expressing PSD-95:mTurq2:mAID (left) and PSD-95:mCitrine (middle) as well as OsTIR1-P2A-mCherry (not shown). B) 5-Ph-IAA induced nearly complete loss of PSD-95:mTurq2:mAID, and increased synaptic levels of PSD-95:mCitrine at the same synapses. Scale bar: 20 µm. C) Changes in PSD-95:mCitrine fluorescence measured at 15-34 synapses of each neuron tracked throughout the experiments (485 in total). Each thin gray line is the average normalized fluorescence measured for the synapses of one neuron (22 neurons from 3 experiments). Thick brown line is the population average. D) PSD-95:mTurq2:mAID fluorescence measured at the same synapses and neurons of C. Thick magenta line is the population average. E) PSD-95:mCitrine fluorescence measured at 14-42 synapses of neurons positive for PSD-95:mCitrine and OsTIR1-P2A-mCherry but negative for PSD-95:mTurq2:mAID (380 in total). Each thin gray line is the average fluorescence measured for the synapses of one neuron (14 neurons from 3 experiments). Thick gray line is the population average. F) Pooled data. Error bars are standard deviations. Test for difference between PSD-95:mTurq2:mAID positive and negative cells – unpaired t-test, without assuming equal variances; applied to data from last time point.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

doi: 10.1101/2024.10.20.619267

Figure Lengend Snippet: Acute elimination of mAID:mTurq2:GKAP is followed by concomitant loss of synaptic PSD-95 and elevated levels of cytosolic PSD-95. A) A rat cortical neuron in culture co-expressing PSD-95:mCit (left) mAID:mTurq2:GKAP (middle) as well as OsTIR1-P2A-mCherry (not shown). B) 5-Ph-IAA induced nearly complete loss of mAID:mTurq2:GKAP, loss of PSD-95:mCit from the same synapses and elevated levels of cytosolic PSD-95:mCit. Scale bar: 20 µm. C) Changes in mAID:mTurq2:GKAP fluorescence measured at 24-78 synapses of each neuron tracked throughout the experiments (1,723 synapses in total). Each thin gray line is the average normalized fluorescence measured for the synapses of one neuron (40 neurons from 5 experiments). Thick brown line is the population average. D) PSD-95:mCit fluorescence measured at the same synapses and neurons of C. Thick magenta line is the population average. E) PSD-95:mCit fluorescence measured at 21-64 synapses of neurons positive for PSD-95:mCit and OsTIR1-P2A-mCherry but negative for mAID:mTurq2:GKAP (752 synapses in total). Each thin gray line is the average fluorescence measured for the synapses of one neuron (18 neurons from 5 experiments). Thick gray line is the population average. F) Changes in cytosolic levels of PSD-95:mCit measured at the cell soma (31 neurons from 5 experiments). Dashed thick magenta line is the population average. G) Changes in cytosolic PSD-95:mCit measured in the cell bodies of neurons positive for PSD-95:mCit and OsTIR1-P2A-mCherry but negative for mAID:mTurq2:GKAP (14 neurons from 5 experiments). Dashed thick gray line is the population average. H) Pooled data. Dashed lines with filled triangles represent cytosolic PSD-95:mCit levels. Error bars are standard deviations. Tests for difference between PSD-95:mTurq2:mAID positive and negative cells – unpaired t-tests, without assuming equal variances; applied to data of last time points.

Article Snippet: Lentiviral vectors for expressing OsTIR1(F74G) in neurons in culture were created as follows: The pAAV-hSyn-OsTIR1(F74G) plasmid described in was obtained from Addgene (Addgene #140730).

Techniques: Expressing, Fluorescence

Journal: Nature Communications

Article Title: Cell-type specific, inducible and acute degradation of targeted protein in mice by two degron systems

doi: 10.1038/s41467-024-54308-9

Figure Lengend Snippet: a Schematic illustration of the hCRBN-S4D and OsTIR1-AID2 systems in mice. The OsTIR1 F74G and hCRBN transgenes are expressed from the Rosa26 locus, under the control of the CAG promoter, and upon loxP-Stop-loxP cassette excision by Cre recombinase. Satb1 V-AID and Satb1 V-S4D were engineered by insertion of mAID and S4D sequences, respectively, at the junction between Venus and Satb1 exon 2 in the Satb1 Venus allele. Immunoblot analysis in total thymocytes ( b ) and flow cytometric analysis in DP thymocytes ( c , d ) after 16 h of intraperitoneal injection with the indicated ligands. Immunoblot and flow cytometric data are representative of three independent biological replicates. Graphs in ( d ) are summarized from three independent biological replicates ( p = 5.373e − 07 for PBS vs 5-Ph-IAA, p = 4.743e − 07 for DMSO vs POM). e , f Kinetics of Satb1 degradation in peripheral blood T cells post-single dose intraperitoneal administration of 5-Ph-IAA or POM, using a standard solvent or a solvent cocktail for improved solubility. Histogram data are representative of three independent biological replicates for OsTIR1-AID2 and hCRBN-S4D (solvent cocktail), and four independent biological replicates for hCRBN-S4D (10% DMSO in PBS). Graphs in ( f ) are summarized from three independent biological replicates for OsTIR1-AID2 and hCRBN-S4D (solvent cocktail), and four independent biological replicates for hCRBN-S4D (10% DMSO in PBS). Data are mean ± s.e.m. Statistical analysis was performed using two-sided unpaired t -test. **** p < 0.00005. Source data are provided as a Source Data file. aa. amino acids, CDS coding sequence, POI protein-of-interest.

Article Snippet: To generate a Rosa26 LSL-OsTIR1 allele, cDNA encoding OsTIR1 F74G was amplified by PCR to introduce AscI sites at the both ends, and inserted into an AscI-cleaved pCTV vector (Addgene, 15912).

Techniques: Control, Western Blot, Injection, Solvent, Solubility, Sequencing

Journal: Nature Communications

Article Title: Cell-type specific, inducible and acute degradation of targeted protein in mice by two degron systems

doi: 10.1038/s41467-024-54308-9

Figure Lengend Snippet: a Summary of protein abundance and fold changes detected by LC–MS in thymus of indicated genotypes and treatments ( n = 3 for each condition). Proteins showing significant changes with adjusted p value < 0.0005 are highlighted in green. b Heatmap displaying protein abundances across different genotypes and treatments. All proteins that were significantly reduced upon 5-Ph-IAA and POM treatment (with adjusted p value < 0.0005) in the thymus of Rosa26 OsTIR1/ + ;Satb1 V-AID/V-AID and Rosa26 hCRBN/+ ;Satb1 V-S4D/V-S4D mice are included. Values were normalized to the mean abundance among samples for each protein. c Venn diagram illustrating shared proteins with significant decrease in abundance across both treatment groups. d The abundances of Satb1, Bcl6, Ikaros (Ikzf1), and Aiolos (Ikzf3) in the thymus of indicated genotypes and treatments. Treatment groups were compared to their respective controls (DMSO-treated Rosa26 hCRBN/+ ;Satb1 V-S4D/V-S4D for the hCRBN-S4D system and PBS-treated Rosa26 OsTIR1/ + ;Satb1 V-AID/V-AID for the OsTIR1-AID2 system). P = 7.79e − 08 and F = 699.5 for Satb1 in the OsTIR1-AID2 system; p = 2.29e − 07 and F = 487.3 for Satb1 in the hCRBN-S4D system; p = 3.04e − 05 and F = 93.13 for Bcl6 in the OsTIR1-AID2 system; p = 0.00138 and F = 23.92 for Bcl6 in the hCRBN-S4D system; p = 0.00458 and F = 15.07 for Ikzf1 in the OsTIR1-AID2 system; p = 0.00318 and F = 17.39 for Ikzf1 in the hCRBN-S4D system; p = 0.345 and F = 1.277 for Ikzf3 in the OsTIR1-AID2 system; p = 0.00147 and F = 17.39 for Ikzf1 in the hCRBN-S4D system. Y axis represents relative protein abundance. Data are mean ± s.e.m. Statistical analyses were performed using two-sided empirical Bayes moderated t -statistics ( a ), or two-sided one-way ANOVA followed by Dunnett’s multiple comparisons test ( d ). ** p < 0.005, **** p < 0.00005. Source data are provided as a Source Data file. CR, Rosa26 hCRBN/+ ; Os, Rosa26 OsTIR1/ + ; WT, Satb1 +/+ ; S4, Satb1 V-S4D/V-S4D ; AI, Satb1 V-AID/V-AID ; No, no treatment; PO, POM; IA, 5-Ph-IAA.

Article Snippet: To generate a Rosa26 LSL-OsTIR1 allele, cDNA encoding OsTIR1 F74G was amplified by PCR to introduce AscI sites at the both ends, and inserted into an AscI-cleaved pCTV vector (Addgene, 15912).

Techniques: Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Communications

Article Title: Cell-type specific, inducible and acute degradation of targeted protein in mice by two degron systems

doi: 10.1038/s41467-024-54308-9

Figure Lengend Snippet: a – c , h , i Anti-tumor effect of temporal PD-1 degradation in hematopoietic cells. MC38-inoculated Rosa26 LSL-OsTIR1/+ ;Pdcd1 AID/AID ;VavCre ( b , c ) or Rosa26 LSL-OsTIR1/+ ;Pdcd1 AID/AID ;E8ICre ( h , i ) mice were intraperitoneally (i.p.) injected with PBS or 5-Ph-IAA every 2 days, starting from 10 days after tumor inoculation. Tumor volume was assessed at specified time points ( b , h ; n = 7 for PBS group and n = 6 for 5-Ph-IAA group for VavCre ; n = 11 for PBS group and n = 10 for 5-Ph-IAA group; p = 0.0104 and 0.01433 for day 20 and 22 for VavCre ; p = 0.03241 and 0.01181 for E8ICre ), and tumor weight was measured after 25 days, post-tumor inoculation ( c , i ; n = 4 each for VavCre and n = 5 each for E8ICre ; p = 7.028e − 05 for VavCre and p = 0.01037 for E8ICre ). d Proportion of PD-1 + cells within CD4 + conventional T cells (Tconv, Foxp3 − ) and Treg (Foxp3 + ) cells (left) and CD8 + T cells (right) was examined after 25 days of MC38 inoculation in Rosa26 LSL-OsTIR1/+ ;Pdcd1 AID/AID ;VavCre mice ( n = 4 for each group, p = 0.008301, 1.033e − 05, and 0.03902 for CD4 + Tconv, Treg, and CD8 + T cells, respectively). e PD-1 expression in CD4 + and CD8 + T cells within tumor was examined after 25 days post-MC38 inoculation. Data are representative of n = 4 each (PBS and 5-Ph-IAA) for Rosa26 LSL-OsTIR1/+ ;Pdcd1 AID/AID ;VavCre and n = 5 each (PBS and 5-Ph-IAA) for Rosa26 LSL-OsTIR1/+ ;Pdcd1 AID/AID ;E8ICre mice. f Fraction of Granzyme B + and IFNγ + cells within CD8+ tumor-infiltrating lymphocytes (TILs) in Rosa26 LSL-OsTIR1/+ ; Pdcd1 AID/AID ;VavCre mice 25 days, post-MC38 inoculation ( n = 4 for each group, p = 0.01073 and 0.01312 for Granzyme B and IFNγ, respectively). g PD-1 and Foxp3 expression in CD4 + T cells within tumor was examined after 25 days, post-MC38 inoculation. Data are representative of n = 4 each (PBS and 5-Ph-IAA) for Rosa26 LSL-OsTIR1/+ ;Pdcd1 AID/AID ;VavCre and n = 5 each (PBS and 5-Ph-IAA) for Rosa26 LSL-OsTIR1/+ ; Pdcd1 AID/AID ;E8ICre mice. Data are mean ± s.e.m. Statistical analysis was performed using two-sided unpaired t -test. * p < 0.05, **** p < 0.00005. Source data are provided as a Source Data file.

Article Snippet: To generate a Rosa26 LSL-OsTIR1 allele, cDNA encoding OsTIR1 F74G was amplified by PCR to introduce AscI sites at the both ends, and inserted into an AscI-cleaved pCTV vector (Addgene, 15912).

Techniques: Injection, Expressing

Journal: Nature Communications

Article Title: Cell-type specific, inducible and acute degradation of targeted protein in mice by two degron systems

doi: 10.1038/s41467-024-54308-9

Figure Lengend Snippet: a Expression of Bcl11b in total thymocytes analyzed by flow cytometry 16 h after a single-dose intraperitoneal injection of PBS or 5-Ph-IAA. Data are representative of three independent experiments. b Thymocytes were analyzed by flow cytometry after 1, 2, and 3 days of daily 5-Ph-IAA treatment or 3-day treatment of PBS. Data are representative of three independent experiments. A schematic diagram for gating the indicated thymocyte populations is shown at the bottom. c Flow cytometric analysis of thymocytes in mice treated with 5-Ph-IAA for 3 days. Data are representative of three independent experiments. d – f Pregnant Rosa26 LSL-OsTIR1/ LSL-OsTIR1 ;Bcl11b AID/AID female mice mated with Bcl11b AID/AID ;VavCre male mice received intraperitoneal injection of 5-Ph-IAA at 13.5 and 14.5 days of gestation ( d ). The total lung ILC2 (CD45 + CD3 – Thy1.2 + ST2 + ) numbers were examined in the offspring at 3 weeks ( e ) and 9 weeks ( f ), by flow cytometry ( n = 3 for each group; p = 0.02488 and 0.0005357 for ST2 + and ST2 + KLRG1 + ILC2 at 3 weeks). Data are mean ± s.e.m. Statistical analysis was performed using two-sided unpaired t -test. * p < 0.05, ** p < 0.005. Source data are provided as a Source Data file.

Article Snippet: To generate a Rosa26 LSL-OsTIR1 allele, cDNA encoding OsTIR1 F74G was amplified by PCR to introduce AscI sites at the both ends, and inserted into an AscI-cleaved pCTV vector (Addgene, 15912).

Techniques: Expressing, Flow Cytometry, Injection