CORRECTION NOTICE Nat. Neurosci.; doi:1.138/nn.3351 mtorc2 controls actin polymerization required for consolidation of long-term memory Wei Huang, Ping Jun Zhu, Shixing Zhang, Hongyi Zhou, Loredana Stoica, Mauricio aliano, Krešimir Krnjević, regg Roman & Mauro Costa-Mattioli In the version of this supplementary file originally posted online, in Supplementary igure 11, the wrong images were provided for total S6K for ig. 1a, β-actin for igures 2a and 3a, actin (bottom) for ig. 5a and p-akt for igure 6a, and the panels corresponding to igures 6a, 6b, 6d and 6e were mislabeled 5b, 5c, 5d and 5e, respectively. The errors have been corrected in this file as of 1 March 21. nature neuroscience
Supplementary Information mtorc2 controls actin polymerization required for consolidation of long-term memory Wei Huang 1,2#, Ping Jun Zhu 1,2#, Shixing Zhang 3,4, Hongyi Zhou 1,2, Loredana Stoica 1,5, Mauricio aliano 1, Krešimir Krnjević 6, regg Roman 3,4, Mauro Costa-Mattioli 1,2,5*. 1 Department of Neuroscience, 2 Center on Addiction, Learning & Memory, 5 Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, Texas, 773. 3 Department of Biology and Biochemistry, 4 Biology of Behavior Institute, University of Houston, Houston, Texas, 7724. 6 Department of Physiology, Mcill University, Montreal H3 1Y6, Canada #These authors contributed equally to this work. *Address correspondence to: Mauro Costa-Mattioli, costamat@bcm.edu
a fepsp slope (% change) 1 4X Hz L-LTP +/+;+/+ WT ( ) +/+;Cre/+ Cre ( ) - - 1 2 flox/flox;+/+ loxed ( ) Contextual Time (min) Auditory Percentage freezing b 6 4 2 +/+;+/+ WT ( ) Cre ( ) Naive +/+;Cre/+ flox/flox;+/+ loxed ( ) 24 h c Percentage freezing 8 6 4 2 Pre-CS CS Supplementary igure 1. L-LTP and fear LTM did not differ between WT, CamKII- Cre and floxed mice. a) Similar L-LTP was induced by four tetanic trains in slices from WT ( +/+;+/+, n = 7) αcamkii-cre ( +/+;Cre/+, n = 6) and floxed mice ( flox/flox;+/+, n = 6) (at 22 min (2,17) =.54, p =.95). b) or contextual fear conditioning, freezing times were determined before the conditioning (Naïve, during 2-min period) and then 24 h after training (during a 3-min period). Similar freezing at 24 h reflects normal contextual LTM in WT (n = 7), αcamkii-cre (n = 7) and floxed (n = 7) mice ( (2,19) =.241, p =.79). c) or auditory fear conditioning, freezing times were measured 24 h post-training either before the onset of the tone (Pre-CS, 2 min) or during the tone presentation (Post-CS, 3 min) (n = 7 per group, (2,19) =.662, p =.94).
a WT fb-ko Nissl b Rictor Synaptophysin PSD95 AD67 Control fb-ko Relative protein level (arbituary unit) 1 β-actin Synaptophysin PSD95 Control fb-ko AD67 Supplementary igure 2. Lack of Rictor does not alter gross brain morphology or synaptic markers. a) Nissl-stained hippocampal sections from WT and fb-ko mice. b) Antibodies against synaptophysin (t =.152; p =.885), PSD95 (t =.147; p =.889) and AD67 (t =.289; p =.784) showed no difference in CA1 between WT (n = 3) and fb-ko mice (n = 4).
mtorc2 a lutamate APV NBQX Total-Akt - + + + - - - + + - b NMDA MK81 - + + + - c BDN TrkB-c - + + + - d Immunoreactivity (% control) β actin 2 * ns * e 3 2 * ns f 3 2 * ns Supplementary igure 3. lutamate, NMDA and BDN activate mtorc2. a-c) Western blots show that, in CA1, glutamate ( μμ, a; (3, 12) = 5.832, * p <.5), NMDA ( μμ, b; (2, 12) = 1.8, * p <.5) or BDN ( ng/ml, c; (2, 6) = 5.493, * p <.5) increased mtorc2-mediated Akt-Ser473 phosphorylation. The results, including effects of selective antagonists APV, MK81 and NBQX (for glutamate), and TrkB-c (for BDN), are summarized in d-f. All comparisons are made with control (untreated) group. APV (aminophosphonovalerate) and MK81 block NMDA receptors; BNQX (dihydroxy-nitro-sulfamoyl-benzoquinoxaline-dione) blocks AMPA-type glutamate receptors. ns = no significance.
a Afferent volley (mv) 2 1 Control (n = 26) fb-ko (n = 18) 2 4 6 8 1 b fepsp slope (v/s) 1 8 6 4 2 _ 2 1 2 3 4 Stimulation intensity (V) Afferent volley (mv) c Paired pulse ratio 1.5 1..5 2 3 4 Interpulse intervals (ms) Supplementary igure 4. Basal synaptic transmission in CA1 does not differ between slices from control and fb-ko mice. a) Plots of presynaptic fiber volley as function of stimulus intensity show no difference in fiber excitability between control (n = 26) and fb-ko mice (n = 21). The data were fitted by linear regressions: R 2 =.991 for controls and.9838 for fb-ko mice. b) Input-output plots show similar EPSPs as function of presynaptic fiber volley amplitude over a wide range of stimulus intensities. The corresponding linear regressions are: R 2 =.6983 for controls and.7392 for fb-ko mice. c) Paired-pulse facilitation of fepsps also did not differ between WT and fb-ko mice, as shown by the plots of the PP ratio (fepsp 2 / fepsp 1 ) for various intervals of paired stimulation.
Visible-platform Latency (s) 4 3 2 Control fb-ko 1 1 2 3 Days Supplementary igure 5. fb-ko mice show normal visuo-motor function. In the visible platform version of the Morris water maze, latencies of escape did not differ between controls and fb-ko mice.
a fepsp slope (% change) Vehicle (n=5) JPK (n=5) 2 mv 5 ms JPK ( nm) fb-ko 2 4 6 8 Time (min) b fepsp slope (% change) Vehicle Cyto-D 2 1X Hz 1 Cyto-D ( nm) _ WT _ 1 2 Time (min) 5 ms 2 mv Supplementary igure 6. Control tests of actin polymerization promoter jasplakinolide (JPK) and inhibitor cytochalasin-d (Cyt-D). a) In slices from fb- KO mice, JPK ( nm) had no effect on basal synaptic transmission expressed by fepsps (n = 5 per group, t = 1.81; p =.12). b) In WT slices Cyt-D ( nm) did not affect early-ltp induced by a single high frequency train ( Hz, 1 s). or these plots, n = 5 for controls (vehicle) and 6 for Cyt-D: at 2 min ( (1, 1) =.42, p =.993). Inset: averaged traces were obtained before and after prolonged application of JPK (a) or before and after 2 min after tetanus (b).
Control Vehicle JPK ( ng) fb-ko -1.94 mm -1.94 mm -2.6 mm -2.6 mm -2.18 mm -2.18 mm -2.3 mm -2.3 mm -2.46 mm -2.46 mm Supplementary igure 7. Sites of JPK infusion into dorsal hippocampus at five rostrocaudal planes. Coordinates are posterior to bregma and cannula tip placements are from mice infused with JPK (filled squares) and vehicle (filled circles). Below are photomicrographs showing representative cannula tracks into the dorsal hippocampus.
a actin Vehicle JPK Vehicle JPK actin -actin/-actin ratio (arbituary unit) 3 2 1 * Vehicle 3 2 1 fb-ko JPK Vehicle JPK WT b 6 Auditory Percentage freezing 4 2 Vehicle JPK Pre-CS CS WT Supplementary igure 8. A low dose of JPK infused into the hippocampus promotes actin polymerization in fb-ko mice. a) Western blots show that thirty min after infusion of JPK ( ng/ml) in the dorsal hippocampus from fb-ko mice the -actin/-actin ratio was increased (at left; n = 4, t = 2.942; * p <.5), but not in the dorsal hippocampus from WT mice (at right; n = 3, t = 1.57; p =.187). Normalized data are shown below. b) Intra-hippocampal infusion of JPK immediately after a weak training (a single pairing of a tone with a 1 s,.7 ma foot-shock) had no effect on auditory fear LTM (n = 15 for vehicle and n = 16 for JPK; H =.768, ANOVA on Ranks, p =.78)
a fepsp slope (% change) 1 1X Hz _ JPK nm Aniso 4 µμ WT _ 1 2 Time (min) JPK (n=8) JPK + Aniso (n=6) b fepsp slope (% change) 1 4X Hz Aniso (n=4) JPK + Aniso (n=5) Aniso 4 µμ _ JPK nm WT _ 1 2 Time (min) Supplementary igure 9. Late-LTP induced by a single tetanic stimulation in combination with JPK is dependent on mrna translation. a) The facilitated L-LTP induced by a single tetanus in the presence of JPK (nm) was suppressed by anisomycin (Aniso 4 µm, at 18 min: 79 ± 12.7% for JPK and 17 ± 4.1% for JPK + Aniso, H = 9.6, p <.1). b) JPK ( nm) had no effect on the impaired L-LTP induced by four tetanic trains in the presence of anisomycin (4 µm, at 22 min: 27 ± 8.6% for Aniso and 33 ± 11.4% for JPK + Aniso, (1, 7) =.5, p =.945).
mtorc2 p-pak (Ser198/23) Total PAK β-actin Vehicle A-443654 Immunoreactivity ( % control) p-pak (Ser198/23) 3 ** 2 ** 2 Vehicle A-443654 Vehicle A-443654 Supplementary igure 1. A-443654 promotes mtorc2 activity in the hippocampus in vivo. Western blots show that 3 min after systemic injection of A-443654 (2.5 mg/kg, i.p.) mtorc2-mediated phosphorylation of both Akt (Ser473) and PAK (Ser198/23) were increased in dorsal hippocampus (bottom left, n = 3 per group, t = 5.726, ** p <.1; bottom right, n = 3 per group, t = 8.263; ** p <.1).
ig. 1a 2 1 Rictor ig. 1c 2 1 Rictor 2 Rictor ig. 1b p-s6k (Thr389) p-s6k (Thr389) Total S6K ig. 1f Total S6K p-s6k (Thr389) Total S6K Control 4 x Hz ig. 1h ig. 1e Control 1 x Hz p-pak (Ser198/23) ig. 2a Time after training (min) 15 3 6 ig. 2c Total PAK Control 4 x Hz Time after training (min) 15 3 6 H om C ec on ag Sh text e oc -al k- on al e on e Supplementary igure 11. Images of full length blots presented in the main figures.
r 1 ig. 4a ri c p-dakt (Ser5) ig. 4c Control fb-ko Actin Pull down ig. 4e ig. 4i Lysate IP: lag DH PDZ L DH PDZ L IP: lag *Heavy chain DH PDZ L DH PDZ L 15 lag lag *Heavy chain myc 2 1 ig. 4j lysate ig. 6a Actin Ve hi A- cle 44 36 54 IP: myc DH PDZ L lag WT Vehicle JPK * p-pak (Ser198/23) ig. 6d Vehicle A-443654 Actin ig. 6e Vehicle A-443654 Actin p-pak (Ser198/23) Total PAK Actin ig. 6b Ve hi A- cle 44 36 54 Ve hi A- cle 44 36 54 fb-ko Vehicle JPK 2 1 Rictor 2 1 15 ig. 5a Lysate 2 2 1 25 2 IP LysTiam1 Ig Tiam1 lag Total Cofilin ig. 4k IP: myc DH PDZ L p-cofilin (Ser3) ig. 4j 2 1 Total PAK 25 2 Cdc42 myc p-pak (Ser198/23) Lysate Control fb-ko Control fb-ko 25 2 Total dakt Control fb-ko 25 Rac1 2 Rac1 Lysate Pull down Control fb-ko Ve hi A- cle 44 36 54 C an to to n -S ig. 3a Total PAK Supplementary igure 11(continued). Images of full length blots presented in the main figures.