EMBO/soda-nel huggingface dataset:
Brassinosteroid signals cooperate with microtubule severing to control filament elongation (2022)
Figure 5

Figure 5 BZR1 binds to the promoter region of KTN80.4 and directly activates its activity

Schematic representation of the KTN80.4 promoter showing the positions of two motifs, BRRE and E-box motifs, which have affinity for the BZR1 family transcription factor (BFTF). ATG denotes the translation start site. TAA denotes the translation stop site. Red arrow indicates E-box motifs of P7 region, brown lines indicate motif sites, gray rectangles indicate introns. Fourteen PCR fragments (P1-P14) were designed for the ChIP analysis.

ChIP analysis of BZR1 binding to the KTN80.4 promoter region in wild type and pBZR1::BZR1-YFP plants. Flowers at stages 13 and 14 were harvested for ChIP analysis performed using the anti-GFP antibody. The red rectangle indicates the P7 region showing the highest enrichment. ACT2 was used as an internal control in PCR quantification. The amounts of DNA fragments were normalized against the input data. Error bars denote the SD of three biological replicates. Student's t test, **P < 0.01, *P < 0.05. Wild type versus pBZR1::BZR1-YFP in P5, **P < 0.0001; wild type versus pBZR1::BZR1-YFP in P6, **P < 0.0001; wild type versus pBZR1::BZR1-YFP in P7, **P < 0.0001; wild type versus pBZR1::BZR1-YFP in P8, **P < 0.0001; wild type versus pBZR1::BZR1-YFP in P9, *P = 0.0167; wild type versus pBZR1::BZR1-YFP in P13, *P = 0.0405.

Activation of KTN80.4 by BZR1. The proKTN80.4::LUC and mproKTN80.4:: LUC constructs were transformed into Nicotiana benthamiana leaves alongside either a pro35S::4xmyc or pro35S::BZR1-4xmyc.

EMSA showing that BZR1 binds to the E-box of the KTN80.4 promoter regions in vitro. Nonlabelled (free) native probe in 10- to 100-fold molar excess relative to the biotin-labeled native probe was used as a cold competitor. The mutant probe was used as a negative control.

Quantitative analysis of the luminescence intensity in (C). Similar results were observed in three biological replicates. Error bars indicate SD. Student's t test, **P < 0.01. pro35S::4xmyc in proKTN80.4::LUC versus pro35S::BZR1-4xmyc in proKTN80.4::LUC, **P < 0.0001.
Brassinosteroid signals cooperate with microtubule severing to control filament elongation (2022)
Figure 6

Figure 6 The qui-2 has reduced microtubule severing and katanin residency

(A-C) Time-lapse images showing that KTN1 is associated with microtubule severing at crossover sites in (A) wild type and (B and C) qui-2 filament cells. GFP-labeled KTN1 fluorescent particles are pseudo-colored in red, and microtubules (labeled with mCherry-TUB6) are shown in green. Yellow arrows indicate KTN1 at the microtubule crossover sites. Asterisks indicate the location at which a microtubule has been severed at a crossover site in A and B, or the location at which a microtubule has not been severed at a crossover site in C. Scale bar, 5 μm in A, B and C.

Katanin localization frequency on microtubules (events per 100 μm2 per second). Purple indicates katanin at microtubule lateral lattice. Green indicates katanin on microtubule crossovers. Orange indicates katanin on microtubule nucleation sites. 440 katanin localization events were detected in area of 2466 μm2 from five cells in wild type. 418 katanin localization events were detected in area of 1720.57 μm2 from four cells in qui-2. The data represent means SD. Student's t test, **P < 0.01. Wild type versus qui-2 in katanin at microtubule lateral lattice, **P = 0.0018; wild type versus qui-2 in katanin on microtubule crossovers, P = 0.5374; wild type versus qui-2 in katanin on microtubule nucleation sites, **P = 0.0032.

Normalized severing frequency (severing events per 100 μm2 per second per crossover) at crossovers were calculated in (D). Error bars indicate SD. Student's t test, **P < 0.01. Wild type versus qui-2, **P = 0.0011

Comparison of katanin recruitment on microtubule crossovers. Green parts indicate the microtubule crossovers without katanin fluorescent particles during observation. Orange parts indicate microtubule crossovers associated with katanin fluorescent particles during observation. A total of 208 microtubule crossovers from wild type (observed area, 2377.45 μm2) and 107 microtubule crossovers from qui-2 (observed area, 1175.38 μm2) were taken into count, respectively.

Comparison of the residency times of katanin in different microtubule positions. A total of 415 katanin localization events from five cells of wild type and 309 katanin localization events from three cells of qui-2 were analyzed. Error bars indicate SEM. Student's t test, **P < 0.01. Wild type versus qui-2 in inactive katanin fluorescent particles at microtubule crossovers, **P < 0.0001; wild type versus qui-2 in inactive katanin fluorescent particles at microtubule nucleation sites, **P < 0.0001.
Bur1 functions with TORC1 for vacuole-mediated cell cycle progression (2022)
Figure 1

Figure 1. Mutation of BUR1 combined with mutations in vacuole inheritance exhibit synthetic growth defects.

(A) Schematic of the synthetic lethal screen used in this study. EMS treated vac17∆ cells which have a plasmid expressing yEmRFP and VAC17, exhibit a red color colony due to over-expression of the yEmRFP protein. Colonies that retain the red color are likely those that cannot lose the plasmid on non-selective medium, and therefore indicate candidate mutations which are synthetically lethal with the vac17∆ mutant.

(B) Schematic of Bur1 protein. Gray box indicates the kinase domain. Blue shaded box; C-terminal tail which is deleted in Bur1-∆C. Arrow; mutation site of bur1-267.

(C) Amino acid alignment by Clustal omega of several CDK kinases in budding yeast. The C-terminal region of Bur1, amino acid residues 373-657 (blue), comprises an unconventional long tail. This region was deleted in the Bur1-∆C mutant used in this study. In addition, this region along with several additional amino acids is missing in the bur1-267 mutant obtained in this study, where W267 (red residue) is substituted with a stop codon. Asterisk; fully conserved residues, colon; strong similarity in amino acid properties, period; weak similarity.

(D) The bur1-267 mutant exhibits a synthetic growth defect with vac17Δ. Haploid colonies from tetrads derived from heterozygous diploids of VAC17/vac17Δ BUR1/bur1-267 were arrayed vertically on YPD (rich medium) plates incubated at 24 ̊C for 3 days. vac17Δ = 17Δ; bur1-267 = b1; bur1-267 vac17Δ double mutant = b1,Δ are indicated.

(E) Quantification of colony size in tetrad dissection, relative to the average of WT colonies. A total of 32 full tetrads were analyzed. Statistical significance (p-value) determined with a one-way ANOVA and Tukey post hoc test. Not a significant difference; ns, p-value > 0.10. **** (p-value < 1 x 10-4). Red bar; average in each category. Error bar; standard deviation (SD).

(F, G) The bur1-ΔC mutant exhibits a synthetic growth defect with vac17Δ. Quantification of colony size in tetrad dissection, relative to the average of WT colonies. A total of 25 full tetrads were analyzed. p-value determined with a one-way ANOVA and Tukey post hoc test. ** (p-value < 1 x 10-2), *** (p-value < 1 x 10-3). Red bar; average in each category. Error bar; standard deviation (SD).

(H) The bur1-ΔC mutant exhibits a synthetic growth defect with the vac17Δ mutant. Plasmids were transformed into a bur1Δ or bur1Δ vac17Δ mutant containing YCp50 [URA3] BUR1. Plasmids tested were pRS415 [LEU2] (mock), pRS415 BUR1, or pRS415 bur1-ΔC. Transformed colonies were cultured in liquid medium and serial dilutions spotted onto SC+5-FOA or SC-Leu-Ura plates. Plates were incubated at 24 ̊C for 3 days.
Bur1 functions with TORC1 for vacuole-mediated cell cycle progression (2022)
Figure 2

Figure 2. A double mutation of BUR1/CDK with a vacuole inheritance mutant or a bur1-267 mutant alone, exhibits a cell cycle delay at G1 phase.

(A, B) The bur1-ΔC vac17Δ double mutant exhibits a delay in progression through G1 phase of the cell cycle. Yeast were grown in YPD medium and collected in log phase growth. DNA content was measured using propidium iodide (PI) staining and assessed by flow cytometry. p-value determined with a one-way ANOVA and Tukey post hoc test. ** (p-value < 1 x 10-2). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment.

(C, D) A bur1-267 mutant also exhibits a delay in progression through G1 phase of the cell cycle. DNA content was measured using PI staining and assessed by flow cytometry. p-value determined with a one-way ANOVA and Tukey post hoc test. **** (p-value < 1 x 10-4). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment.

(E, F) The bur1-ΔC vac17Δ double mutant shows accumulation of Whi5 in the nucleus. WT, vac17Δ, bur1-ΔC, and bur1-ΔC vac17Δ cells, which express Whi5-3xGFP and Nup188-mCherry from endogenous loci, were incubated at 24 ̊C. Cells were scored for the presence of Whi5-3xGFP in within the nucleus (Nup188-mCherry). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment. p-value determined with a one-way ANOVA and Tukey post hoc test. ** (p-value < 1 x 10-2). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment. Scale bar, 2 μm.
Bur1 functions with TORC1 for vacuole-mediated cell cycle progression (2022)
Figure 3

Figure 3. The double mutation of BUR1/CDK with a vacuole inheritance mutant is not defective in de novo synthesis of the vacuole

(A) The bur1-∆C mutant, but not bur1-∆C vac17∆ double mutant, shows normal vacuole inheritance. Quantitative analysis of vacuole inheritance in wild-type, vac17∆, bur1-∆C, and bur1-∆C vac17∆ cells. Cells were pulse-chase labeled with FM4-64, and vacuole inheritance was assessed as the percent cells with inherited vacuoles. Error bars; SD calculated from three independent experiments with at least 100 cells counted in each strain/experiment.

(B) The bur1-∆C vac17∆ double mutant cells have the ability to generate a vacuole de novo, which suggests that the cell-cycle delay is due to a defect in Bur1-dependent signaling. WT, vac17Δ, bur1-∆C, and bur1-∆C vac17∆ cells which express Vph1-GFP from its endogenous locus, were pulse labeled with the vacuole specific dye FM4-64. WT and bur1-∆C cells have both FM4-64 and Vph1-GFP signals in both mother and daughter cells. vac17Δ and bur1-∆C vac17∆ cells have both Vph1-GFP and FM4-64 on the vacuole in mother cells, however the daughter cells solely have a Vph1-GFP labeled vacuole, indicating that the vacuole was generated de novo, rather than inherited from the mother vacuole. Arrowheads; new vacuoles in daughter cells. Scale bar, 2 μm.
Bur1 functions with TORC1 for vacuole-mediated cell cycle progression (2022)
Figure 4

Figure 4. BUR1 functions in parallel with the TORC1 pathway.

(A) In vivo phosphorylation of Sch9 is reduced in bur1-∆C vac17∆ cells. WT, bur1-∆C, vac17∆, or bur1-∆C vac17∆ cells expressing SCH9-HA from the URA3 locus were grown in YPD. Cell lysates were analyzed by immunoblot with antibodies directed against HA (top) and Pgk1 (loading control).

(B) In vivo phosphorylation of Sch9 is reduced in bur1-267 cells. WT, vac17∆, bur1-267, or bur1-267 vac17∆ cells expressing SCH9-HA from the URA3 locus were grown in YPD. Cell lysates were analyzed by immunoblot with antibodies directed against HA (top) and Pgk1 (loading control).

(C) bur1-∆C shows high rapamycin sensitivity. WT, tor1∆, bur1-∆C, tor1∆ bur1-∆C or vac17∆ cells were cultured in liquid YPD medium and serial dilutions were spotted onto YPD plates with 0, 2, 4 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days.
Bur1 functions with TORC1 for vacuole-mediated cell cycle progression (2022)
Figure 5

Figure 5. Bur1 phosphorylates Sch9 in vivo and in vitro.

(A) Western blot analysis of immunoprecipitated Bur1-HA or Bur1-∆C-HA used in (B).

(B) In vitro, Bur1 directly phosphorylates the kinase domain of Sch9 as well as Bur1 itself. pRS425 BUR1-HA and pVT102-Ura BUR2, the essential cyclin for BUR1, were co-expressed in a bur1Δ mutant, immunoprecipitated using anti-HA antibody and protein A beads, and then incubated with [γ-32P]ATP and GST, GST-N-Sch9 (1-390) or GST-C-Sch9 (391-824). Proteins were eluted with SDS sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes exposed to x-ray film (top) or stained with Ponceau S (bottom). Red arrowhead; phosphorylated GST-C-Sch9. Gray arrowhead; auto-phosphorylated Bur1.

(C) Phosphorylation sites determined by mass spectrometry in Sch9 are indicated (red lettering). Green circle; Pkh target site (T570), blue circles; main TORC1 target sites (T723, S726, T737, S758, and S765).

(D) Sch9-2D3E, but not Sch9-3A-2D3E, partially suppressed the rapamycin sensitivity of tor1∆ cells. tor1∆ which expresses pRS426 (mock), pRS425 TOR1, pVT102-Ura (mock), pVT102-Ura Sch9, pVT102-Ura Sch9-2D3E, or pVT102-Ura Sch9-3A-2D3E, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2-3 days.

(E) The Sch9-2D3E mutant did not suppress the rapamycin sensitivity of bur1-∆C cells. bur1-∆C which expresses pRS426 (mock), pRS426 BUR1, pVT102-Ura (mock), pVT102-Ura SCH9, pVT102-Ura sch9-2D3E, pVT102-Ura sch9-3A-2D3E, pVT102-Ura sch9-3D/E, or pVT102-Ura sch9-3D/E-2D3E were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 3 days.
Bur1 functions with TORC1 for vacuole-mediated cell cycle progression (2022)
Figure 6

Figure 6. Bur1 function in the nucleus is required for TORC1 signaling.

(A) Bur1 fused SV40-NLS does not rescue the rapamycin sensitivity of bur1-∆C compared to Bur1 without the NLS. bur1-∆C which expresses pRS416 (mock), pRS416 BUR1, pRS416 BUR1-mNG, pRS416 BUR1-mNG-NLS, pRS416 bur1-∆C, pRS416 bur1-∆C-mNG-NLS or pRS416 bur1-∆N, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0, 4, 8, or 16 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days.

(B) Adding nuclear-export signal (NES) of PKI to Bur1 and/or deletion of the importin α-dependent NLS from Bur1 resulted in higher sensitivity to rapamycin compared to Bur1 without this NES. bur1-∆C which expresses pRS426 (mock), pRS426 BUR1, pRS426 bur1-∆N, pRS426 BUR1-mNG, pRS426 BUR1-mNG-NES, or pRS426 bur1-∆N-mNG-NES, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0, 4, 8, or 16 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days.

(C) Model for the roles of Bur1, TORC1 and Sch9 for progression through G1 phase of the cell-cycle. Bur1 and TORC1 act in parallel to phosphorylate Sch9. We propose that similar to TORC1, Bur1 phosphorylates Sch9 on the vacuole membrane, or alternatively that Sch9 which is phosphorylated by TORC1 on the vacuole, translocates to the nucleus where it is further phosphorylated by Bur1. Then Sch9 acts in parallel with Cdc28/Cdk1-Cln3, in a process that results in the export of Whi5 from the nucleus and cell-cycle progression through early G1 phase.

Expanded View Figure Legends
Ca2+/Calmodulin Binding to PSD-95 Mediates Homeostatic Synaptic Scaling Down (2017)
Figure 1

Figure 1. NMR analysis of Ca2+/CaM binding to PSD-95_pT19.

(A) Overlay of 15N-1H HSQC spectra of 15N-labeled Ca2+/CaM by itself (red peaks) and after addition of saturating, unlabeled PSD-95 (1-21) (blue peaks) or saturating, unlabeled PSD-95_pT19 (green peaks). All NMR experiments were recorded at 600 MHz 1H frequency.

(B) Chemical shift perturbation of CaM amide resonances in the absence vs. presence of PSD-95_pT19. Chemical shift difference (CSD) plotted on the vertical axis was defined as CSD = {(HNA - HNB)2 + (15NA - 15NB)2}1/2 , where "A" and "B" designate free and bound states of CaM. HN and 15N represent amide 1H and 15N chemical shifts, respectively. Perturbations greater than the average were used as active ambiguous restraints in the Haddock docking calculation (see Methods).

(C) Representative data from IPAP [15N-1H]-HSQC spectra of 15N-labeled Ca2+/CaM bound to PSD-95_pT19 in the absence (blue) and presence of 17 mg/ml filamentous bacteriophage Pf1 (red). Residual dipolar couplings (RDCs) for backbone HN resonances are calculated (in Hertz) as the difference in splitting for the 15N-{1H} doublet components (red peaks) relative to the isotropic 1JNH coupling (blue peaks). RDC values are calculated to be +40, +30, +20, +20, and -20 Hz for I100, I27, N137, D64, and I130, respectively.

(D) Correlation between observed RDCs (panel C) versus RDCs calculated from the final NMR derived structure of Ca2+/CaM bound to PSD-95_pT19. The correlation coefficient (R) equals 0.99 and Q is 0.11.
Ca2+/Calmodulin Binding to PSD-95 Mediates Homeostatic Synaptic Scaling Down (2017)
Figure 2

Figure 2. Ca2+/CaM makes electrostatic contacts with PSD-95 phosphorylated at T19.

(A) NMR structure of Ca2+/CaM (grey) bound to unphosphorylated PSD-95. PSD-95 residues D2 (depicted as starting point) to R13 are resolved and form an -helix (cyan) (Zhang et al., 2014). Residues 14-19 of unphosphorylated PSD-95 are unstructured and not shown. Ca2+ ions bound to CaM are depicted by orange spheres.

(B) NMR structure of Ca2+/CaM bound to PSD-95 phosphorylated at T19 (PDB accession no. 5J7J). Residues 14-19 of phosphorylated PSD-95 adopt a hook-like structure (highlighted in red) not seen in unphosphorylated PSD-95. This hook-like structure is stabilized by PSD-95 residues E17 and pT19 forming salt bridges with R126 and K115, respectively. Side-chain atoms are depicted by a stick representation for PSD-95 residues Q15, E17 and T19 (red) and CaM residues K115, T117, and R126 (blue).

(C) Intermolecular contacts observed for residues in the helical segment of PSD-95_pT19 (side-chain atoms shown as stick representation) and residues in CaM (represented by the colored ovals).
Ca2+/Calmodulin Binding to PSD-95 Mediates Homeostatic Synaptic Scaling Down (2017)
Figure 3

Figure 3. PSD-95 peptide titration with Ca2+/CaM.

(A) FP titrations showing the binding of wild-type (WT) CaM to PSD-95 (1-21) peptides, either WT (blue), Q15A (salmon), E17R (black), T19E (green), or T19K (yellow). The solid lines represent optimal theoretical curves calculated using a one-site binding model described in Methods. The calculated dissociation constants in each case are given in Table 1.

(B) FP titrations showing the binding of R126E CaM to PSD-95 peptides, either WT (blue) or E17R (black).

(C) FP titrations showing the binding of K115E CaM to PSD-95 peptides T19K (yellow) or T19E (green).

(A-C) Basal levels of FP obtained in the absence of Ca2+/CaM were determined for each titration and subtracted. Mean values from three independent titrations (+ S.D.) are indicated and fitted to a one-site binding model (see Methods).
Ca2+/Calmodulin Binding to PSD-95 Mediates Homeostatic Synaptic Scaling Down (2017)
Figure 4

Figure 4. Prolonged BIC treatment results in reduction of PSD-95 palmitoylation.

Cultured cortical neurons were treated with BIC (50 μM) at 15 DIV for 48h or at 17 DIV for 2 h or left untreated before harvesting, extraction, and analysis of palmitoylation by the biotin switch method and pull down with NeutrAvidin-agarose beads.

(A) Representative immunoblots of pull down samples (top two panels) and total lysate (bottom two panels) for PSD-95 and, as control, the trimeric G protein subunit G-i3. Omission of NH2OH before biotinylation resulted in no NeutrAvidin pull down as negative control for non-specific pull down.

(B) Quantification of PSD-95 palmitoylation. Seven samples from three independent sets of cultures were analyzed per condition (**p<0.01; t-test).
Ca2+/Calmodulin Binding to PSD-95 Mediates Homeostatic Synaptic Scaling Down (2017)
Figure 5

Figure 5. Point mutations of PSD-95 that disrupt Ca2+/CaM binding prevent BIC-induced loss of surface AMPARs during scaling down.

Cultured hippocampal neurons were infected at 14 DIV with lentivirus for expression of both shRNA against PSD-95 (sh95) and sh-resistant PSD-95-EGFP. Thus endogenous PSD-95 was replaced with either wild-type (WT), E17R, or T19K PSD-95. Cultures were treated with or without BIC (50 μM) at 17 DIV for 24-48 h or left untreated before fixation and staining for surface GluA1.

(A) Representative confocal microscopic images of PSD-95-EGFP fluorescence (green) and surface GluA1 immunostaining (red) at low (top panels; scale bar: 20 µm) and high enlargement (bottom panels; scale bar: 5 µm).

(B) Quantification of surface GluA1 intensity from (A) showed reduction in AMPAR surface levels during scaling down in neurons expressing WT PSD-95 but not in those expressing either E17R or T19K mutant. Three dendritic segments from each of 31-42 neurons from three independent sets of cultures were analyzed per condition (**p<0.01; n.s., not significant; one-way ANOVA followed by Bonferroni's post hoc test).
Ca2+/Calmodulin Binding to PSD-95 Mediates Homeostatic Synaptic Scaling Down (2017)
Figure 6

Figure 6. Point mutations of PSD-95 that disrupt calmodulin binding prevent BIC-induced loss of PSD-95 from spines.

Cultured hippocampal neurons were co-transfected with mCherry and EGFP-tagged PSD-95 wild-type (WT), E17R, or T19K at 10-11 DIV and treated with BIC (50 μM) or left untreated at 17 DIV for 24-48 h before fixation.

(A) Representative confocal microscopic images of PSD-95-EGFP (green) and mCherry (red) used as "fill" to visualize the dendrite with spine heads (scale bar: 2 µm) after control (top) and BIC treatment (bottom). Individual channels are shown in grayscale.

(B) BIC treatment reduced spine enrichment of WT but not E17R or T19K PSD-95 as quantified as the ratio of EGFP intensity in spine heads relative to that in the adjacent dendritic shaft. Values were normalized to control treatments, which were set to equal 100% for each PSD-95 construct.

(C) mCherry showed no enrichment in spines and BIC had no effect on its localization.

(D-F) Spine enrichment and puncta size and intensity of PSD-95 were comparable under basal conditions for WT, E17R, and T19K PSD-95 (A.U., arbitrary unit).

(G-I) Spine density, head diameter, and length were comparable under basal conditions for neurons expressing WT, E17R, and T19K PSD-95.

(B-I) Statistical analysis was performed on n=166-202 spines from 10-16 neurons per condition from three independent sets of cultures (***p<0.001; n.s., not significant; one-way ANOVA followed by Bonferroni's post hoc test).
Ca2+/Calmodulin Binding to PSD-95 Mediates Homeostatic Synaptic Scaling Down (2017)
Figure 7

Figure 7. R126E mutation on CaM restores the BIC-induced decrease in surface GluA1 that is impaired by PSD-95 E17R mutation.

Cultured hippocampal neurons were transfected at 10-11 DIV with the indicated CaM-IRES-ECFP plasmids and infected at 14 DIV with lentivirus expressing shRNA against PSD-95 (sh95) and sh-resistant wild-type (WT) or E17R PSD-95-EGFP. Cultures were treated with BIC (50 μM) or left untreated at 17 DIV for 24-48 h before fixation.

(A) Representative confocal microscopic images of intrinsic ECFP and EGFP fluorescence, and surface GluA1 immunostaining at low (top panels; scale bar: 20 µm) and high enlargement (bottom panels; scale bar: 5 µm).

(B) Quantification of surface GluA1 intensity from (A) showed impaired AMPAR downscaling in neurons expressing PSD-95-E17R in presence of wild-type CaM (CaM-WT) that was rescued by coexpression of CaM-R126E. Three dendritic segments from each of 18-26 neurons from three independent sets of cultures were analyzed per condition (**p<0.01,*p<0.05 vs. untreated basal levels; one-way ANOVA followed by Bonferroni's post hoc test).