Neuron Supplemental Information Neuronal Activity and CaMKII Regulate Kinesin-Mediated Transport of Synaptic AMPARs Frédéric J. Hoerndli, Rui Wang, Jerry E. Mellem, Angy Kallarackal, Penelope J. Brockie, Colin Thacker, David M. Madsen, and Andres V. Maricq
Figure S1. GLR-1 transport is modified by presynaptic glutamate release and postsynaptic VGCCs. Supplemental data associated with Figure 1. (A) (Left) Confocal image of GLR-1::GFP expressed in the AVA neurons. Indicated is the region where GLR-1 transport is monitored (labeled proximal processes) as well as the nerve ring that contains AMPARs activated during voltage-clamp experiments. Scale bar represents 10 m. (Right) Confocal images of GLR-1::GFP in AVA cell bodies and nerve ring processes in transgenic WT and unc-43(lf) mutants. Arrowheads indicate sites where GLR-1::GFP accumulates in unc-43(lf) mutants. Scale bar represents 5 m. (B and C) GLR-1::GFP transport in AVA neurons from eat-4(ky5) loss-of-function mutants (B) and unc-36(rnai) mutants (C), compared to wild-type worms (Control). Scale bars represent 5 m. (D) The total number of transport events normalized to WT controls. n > 1
8 worms per condition. *** p < 0.001, ANOVA with Tuckey s multiple comparison. Error bars indicate SEM. 2
Figure S2. CaMKII is not required for UNC-116/KIF5 movement. Supplemental data associated with Figure 2. (A) Kymographs (left) and quantification (right) of UNC-116::GFP transport in WT and unc-43(lf) mutants normalized to WT. n = 6 worms per genotype. Scale bar represents 5 μm. (B) Quantification of UNC-116::GFP FRAP in WT and unc-43(lf) mutants. n = 6 worms per genotype. (C) Kymographs (top) and quantification (bottom) of EBP-2::GFP polymerization events in WT and unc-43(lf) mutants normalized to WT. n = 8 worms per genotype. (D) VAMP::mCherry transport in WT, unc-43(lf) mutants and unc-116(rh24) mutants (top). Quantification of the total number of VAMP::mCherry transport events 3
(bottom) normalized to WT. n = 11 for WT and unc-43(lf), n = 4 for unc-116(rh24). * p < 0.05. Error bars indicate SEM. 4
Figure S3. ACh-gated currents are not disrupted in unc-43/camkii mutants. Supplemental data associated with Figure 2. Currents evoked by application of 200 M ACh (A) and the peak amplitude of AChgated current (B) in the AVA neurons of wild-type (Control) and unc-43(lf) mutants. n = 5 worms per genotype. Error bars indicate SEM. 5
Figure S4. UNC-43, UNC-116 and GLR-1 co-localize in the AVA cell body and processes. Supplemental data associated with Figure 4. (A and B) Confocal images of UNC-116::mTFP-1, mcherry::unc-43 and GLR-1::GFP in the AVA cell bodies (A) and AVA processes (B). (C) Confocal images of GLR- 1::GFP and UNC-43::mCherry expressed in the AVA processes of unc-116(rh24) lossof-function mutants. Scale bars represent 5 m. 6
Figure S5. KLC-2 co-localizes with GLR-1 at synapses. Supplemental data associated with Figure 4. (A) Confocal images of GLR-1::GFP and KLC-2::TagRFP-T in the AVA processes. (B) Confocal images of wild type and mutant variants of KLC-2 fused to TagRFP-T in the AVA processes. Scale bars represent 5 m. 7
Figure S6. GLR-1 and GLR-2 subunit abundance are altered in unc-43/camkii mutants. Supplemental data associated with Figure 4. (A) Confocal images of SEP::GLR-1 fluorescence (left) and quantification of SEP fluorescence (right). (B) Confocal images (left) and quantification (right) of GLR- 1::mCherry and GFP::GLR-2 fluorescence in the AVA neurons of transgenic WT and unc -43(lf) mutants. (C) Glutamate-gated currents in the AVA neurons of glr-2(lf) and unc-43(lf) mutants with and without expression of a wild-type glr-2 transgene in AVA. * p < 0.05, ** p < 0.01, *** p < 0.001.Scale bars represent 5 m. Error bars indicate SEM. 8
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Figure S7. FRAP of surface GLR-1 proceeds at a different rate than FRAP of total GLR- 1. Supplemental data associated with Figures 5 and 6. (A) Confocal images of SEP and mcherry fluorescence before, immediately after, and 5, 10, and 20 min after photobleaching both SEP and mcherry. (B) The ratio of SEP to mcherry fluorescence at synaptic puncta. n = 4 worms. * p < 0.05. (C) Confocal images (top) and FRAP quantification (bottom) of SEP (left) and mcherry (right) fluorescence in transgenic worms that expressed UNC-116::miniSOG in AVA. Blue bars represent light inactivation (CALI) of UNC-116::miniSOG in the AVA processes. FRAP of mcherry, but not of SEP, in transgenic worms exposed to blue light (CALI) was significantly different from transgenic worms without light exposure. n > 5 per condition. p < 0.05 (ANCOVA analysis). (D) Transport of GLR-1::GFP and mcherry::unc-43 in the AVA processes. White arrowheads indicate mcherry::unc-43 transport events. The merged kymographs show no evidence of co-transport. Scale bars represent 5 m. Error bars indicate SEM. 10
Figure S8. NMDA-gated currents are not appreciably disrupted by CALI inactivation of minisog::unc-43 in AVA. Supplemental data associated with Figure 8. Two examples of NMDA-gated currents in response to 0.5 second pressure applications of 1 mm NMDA at 1 minute intervals both before (black) and during (blue) CALI inactivation of minisog::unc-43. 11
Figure S9. UNC-43/CaMKII is required for GLR-1 transport in adult animals. Supplemental data associated with Figure 9. (A and B) Kymographs (A) and quantification (B) of GLR-1::GFP transport in AVA before (Control) and after heat shock in WT and unc-43(lf) mutants, and transgenic mutants with a Phsp::unc-43 transgene. Control worms were kept at 15 before imaging. Heat shocked worms were transferred from 15 to 31 for 1 hour before imaging. Transport events were normalized to wild-type control worms (WT). n > 8 per condition and genotype. * p < 0.05. Error bars indicate SEM. 12
Supplemental Experimental Procedures Plasmids. The following plasmids were used to generate transgenic animals: pjm23, lin- 15(+); pdm1437, Prig-3::HA::glr-1::gfp; pdm1556, Prig-3::HA::glr-1::mCherry; pdm1633, Pflp-18::unc-116::gfp; pdm1494, Prig-3::mCherry; pdm1550, Prig- 3::HA::glr-1::Dendra2; pdm1983, Prig-3::sep::mCherry::glr-1; pdm1973, Pflp- 18::gfp::glr-2; pdm1772, Pflp-18::mCherry::unc-43; pdm1475, Peat- 4::ChR2::mCherry; pct61, Pegl-20::nls::dsRed; pdm2033, Pflp-18::glr-2; pdm2032, Prig-3::stg-2; pdm1698, Prig-3::sol-1; pdm1509, Prig-3::sol-2; pdm2234, Pflp- 18::miniSOG::unc-43; pdm2235, Pflp-18::unc-116::miniSOG; pdm2241, Pflp- 18::TagRFP-T::mCaMKII; pdm2245, Pflp-18::TagRFP-T::unc-43(K42R); pdm2246, Pflp-18::TagRFP-T::unc-43(K42R; T286D); pdm2162, Pflp- 18::mCherry::miniSOG::unc-43; pdm2166, Pflp-18::unc-116:: minisog::mcherry; pdm2200, Pflp-18::unc-43; pdm2303, Peat-4::HisCl1; pdm2299, Pflp-18::HisCl1; pdm2314, Pflp-18::unc-36(cDNA); pdm2208, Pflp-18::klc-2::TagRFP-T; pdm2326, Pflp-18::klc-2(T208A)::TagRFP-T; pdm2328, Pflp-18::klc-2(S240A)::TagRFP-T; pdm2330, Pflp-18::klc-2(S276A)::TagRFP-T; pdm2336, Pflp-18::klc-2(T208A; S240A; S276A)::TagRFP-T; pdm2337, Pflp-18::klc-2(T208D; S240D; S276D)::TagRFP-T; pdm1466, Pmec-3::VAMP::mCherry; pdm1562, Prig-3::VAMP::mCherry, pct91, Pmec-3::ChIEF; pdm1501, Prig-3::ebp-2::gfp; pdm2307, Prig-3::unc-116::mTFP-1; pdm2198, Phsp 16-2::unc-43, pdam36, Prig-3::ChIEF. All fusion proteins were functional as they rescued the mutant phenotype when expressed in the relevant mutant background. 13
The flp-18 and rig-3 promoter sequences were based on that published in Feinberg et al., 2008. unc-116(rnai) and unc-36(rnai) were expressed in the AVA neurons using the flp-18 promoter sequences and generated using published protocols (Esposito et al., 2007). Primer sequences are available upon request. To identify potential CaMKII phosphorylation sites in KLC-2, amino acid sequences (isoforms A and B) from WormBase (www.wormbase.org) were analyzed using algorithms such as NetPhosK and GPS 2.1. Sites predicted by both NetPhosK and GPS 2.1 were chosen for mutational analysis. Transgenic arrays. In most cases, transgenic worms were generated by germline transformation of lin-15(n765ts) mutants by microinjecting the lin-15(+) rescuing plasmid (pjm23). In cases where lin-15 mutants were not used, transgenic worms were identified by expression of soluble mcherry or dsred under the regulation of a cell specific promoter encoded by the co-injected plasmid. Integrated and extra-chromosomal arrays were: akis141, Prig-3::HA::glr-1::gfp; akis154, Prig-3::HA::glr-1::Dendra2; akis201, Prig-3::sep::mCherry::glr-1; akis222, Prig-3::HA::glr-1::mCherry + Pflp- 18::gfp::glr-2; akex3707, Pflp-18::TagRFP-T::unc-43(K42R); akex3708, Pflp- 18::TagRFP-T::unc-43(K42R); akex3730, Pflp-18::TagRFP-T::unc-43(K42R; T286D); akex2952, Pflp-18::unc-116(RNAi) + Prig-3::mCherry; akex3698, Pflp-18::TagRFP- T::mCaMKII; akex3673, Pflp-18::glr-2 + Prig-3::sol-1 + Prig-3::sol-2 + Prig-3::stg- 2::gfp + Prig-3::mCherry; akex2703, Pflp-18::mCherry::unc-43 + Peat-4::::mCherry; akex3536, Pflp-18::mCherry::miniSOG::unc-43; akex3559, Pflp-18::unc- 116::mCherry::miniSOG; akex1913, Prig-3::HA::glr-1::mCherry + Pflp-18::unc- 14
116::gfp; akex3714, Pflp-18::miniSOG::unc-43; akex3729, Prig-3::HA::glr-1::mCherry + Pflp-18::miniSOG::unc-43; akex4217, Pflp-18::unc-36(cDNA); akex3989, Pflp- 18::HisCl1 + Prig-3::ChIEF + Prig-3::mCherry; akex3687, Pmec3::VAMP::mCherry + Pmec-3::ChIEF; akex4099, Prig-3::ebp-2::gfp; akex4100, Prig-3::ebp-2::gfp; akex4101, Prig-3::ebp-2::gfp, akex4102, Prig-3::ebp-2::gfp; akex1433, Prig- 3::VAMP::mCherry; akex3723, Pflp-18::klc-2::TagRFP-T; akex4235, Pflp-18::klc- 2(S276A)::TagRFP-T; akex4234, Pflp-18::klc-2(S240A)::TagRFP-T; akex3773, Phsp16-2::unc-43; akex3712, Pflp-18::miniSOG::unc-43; akex4047, Pflp-18::unc- 116::miniSOG; akex4033, Pflp-18::glr-2 + Prig-3::mCherry; akex4239, Peat- 4::HisCl1; akex4222, Pflp-18::unc-36(RNAi); akex 4223, Pflp-18::klc- 2(T208A)::TagRFP-T; akex 4224, Pflp-18::klc-2(T208A)::TagRFP-T; akex 4225, Pflp- 18::klc-2(T208A)::TagRFP-T; akex4226; Pflp-18::klc-2(T208D; S240D; S276S)::TagRFP-T; akex4228, Pflp-18::klc-2(T208D; S240D; S276S)::TagRFP-T; akex4231, Prig-3::unc-116::mTFP-1 + Pflp-18::mCherry::unc-43; akex4233, Pflp- 18::klc-2(T208A; S240A; S276A)::TagRFP-T; akex4236, Pflp-18::mCherry::unc-43; akex4240, Pflp-18::klc-2(T208D; S240D; S276S)::TagRFP-T; akex4241, Pflp-18::klc- 2(T208A; S240A; S276A)::TagRFP-T; akex4242, Pflp-18::klc-2(T208A; S240A; S276A)::TagRFP-T. Imaging. For UNC-116::GFP FRAP, worms were mounted as described previously for in vivo microscopy (Hoerndli et al., 2013). The bleached region included the region of interest (ROI) as well as 60 m to the left and right of the ROI. Confocal stacks of the ROI were taken 1, 2, 4, 8 and 16 minutes after photobleaching. For dual streaming 15
imaging of GLR-1::GFP and mcherry::unc-43, imaging was performed as described in Hoerndli et al., 2013. Heat shock. Transgenic wild-type and unc-43(lf) mutants that expressed GLR-1::GFP in AVA, and unc-43(lf) mutants that also expressed Phsp16-2::unc-43 were kept at 15 for at least one generation prior to the experiment. Control worms (no heat shock) were kept at 15 before imaging. Heat shocked worms were transferred from 15 to 31 for one hour and then kept at room temperature for one hour after heat shock and before imaging. Image analysis. For quantification of SEP puncta (Figure S6A and S6B), a linescan measurement was taken on maximal projection images and analyzed using a custom written MatLab algorithm as described in Hoerndli et al., 2013. From this analysis we extracted the total amount of SEP per m. For FRAP experiments shown in Figure 5A and 5B, the distal region of the AVA processes was chosen to minimize the effect of diffusion from the cell body. Single region of interests of 5-6 GLR-1::GFP puncta per animal, were analyzed in ImageJ, corrected for background and used to generate % FRAP shown in Figure 5B.For FRAP of GLR-2::GFP (Figure 5C 5D), the proximal region of the AVA processes was chosen because the fluorescent signal in the distal region of unc-43(lf) mutants was too low to quantify. Because the signal was more diffuse, we traced the AVA region for FRAP using the segmented line tool in ImageJ with a width of 10 pixels, extracted mean fluorescence values for this region and subtracted for background. For GLR-1::Dendra2 photoconversion experiments (Figure 5E and 5F), the 16
fluorescent signal was quantified by drawing ROIs on the maximal projection images of confocal stacks, corrected for background and expressed as a percentage of the signal immediately after photoconversion. This same procedure was used for experiments that combined CALI and photoconversion except that the signal was expressed as a percentage of the signal remaining after CALI (Figure 7C E). For SEP::mCherry::GLR-1 FRAP, maximal projection images were quantified by drawing ROIs around mcherry puncta, subtracting background fluorescence and calculating the percentage of the signal before photobleaching. The same regions were used to quantify SEP FRAP. 17