Myc or HA epitope-tagged Plk2, hyperactivating Plk2 (T236E), kinase-dead Plk2 (K108M), C-terminal Plk2 (aa395–682), polo domain mutant Plk2 (W504F), NSF, GluA2 and SPAR were expressed from pGW1-CMV. The following oligonucleotide (5′ to 3′) was inserted into pLL3.7 vector for Plk2-RNAi: GCATAGGGATCTCAAGCTA. For Plk2 rescue the following silent mutations were made (in bold) in full-length rat Plk2: GCATAGAGACCTCAAGCTA. For yeast-two-hybrid analysis, Plk2 fragments were cloned into pBHA and NSF into pGAD. Plk2 PBind was cloned into pEGFPC1 for GFP fusion protein and pMAL-c2x for MBP fusion. Site-directed mutagenesis was performed using QuikChange Kit (Stratagene) and confirmed by DNA sequencing.
and SPAR antibodies14
have been described. The following antibodies were purchased commercially: mouse NSF (Calbiochem), mouse GluA2 (BD Pharmingen), mouse and rabbit GFP (Qbiogene and Invitrogen), goat Plk2 (SNK C-18, Santa Cruz Biotechnology), rabbit GluA1 (Calbiochem and Millipore), mouse PSD-95 clone K28/43 (NeuroMab), rabbit GRIP1 (Millipore), mouse PICK1 clone L20/8 (NeuroMab), rabbit phospho-Ser880 GluA2 (Millipore). AlexaFluor-488, -555 and -647 (Invitrogen) were used as secondary antibodies.
Glutathione-S-transferase (GST) fused to Plk2c (aa352–682) was purified from E. coli and coupled to glutathione sepharose resin. Adult rat brain lysates were applied to a GST-Plk2c-resin column, washed extensively, and eluted. Eluates were separated on SDS-PAGE gels and visualized by Coomassie blue. Bands were excised in the ~50–120kD range that contained most of the visible proteins, cut into slices, and digested in-gel by trypsin. Peptides were identified by liquid chromatography-tandem mass spectrometry.
Cells were lysed in IP buffer (50mM Tris pH 8.0, 150mM NaCl, 1% NP-40, plus pepstatin, leupeptin, PMSF), centrifuged to remove cell debris, and incubated 3–4 h, 4°C with 5 μg GST-Plk2c (or GST alone). Following wash with GST-lysis buffer (20mM Tris-Cl, pH 8.0, 200mM NaCl, 1mM EDTA, 0.5% Nonidet P-40, 2 μg/ml aprotinin, 1 μg/ml leupeptin, 0.7 μg/ml pepstatin and 25 μg/ml PMSF), bound proteins were analyzed by SDS-PAGE and immunoblotting. For SDS wash, GST-Plk2c coupled resin was incubated in 1% SDS for 1 min, then quenched by 10-fold dilution into IP buffer.
In vitro binding and ATPase Assay
His6-NSF was purified from E. coli by disruption in lysis buffer (100mM Hepes/KOH, pH 7.4, 500mM KCl, 5mM MgCl2, 1mM DTT, and 0.5mM ATP plus protease inhibitors), incubation with Ni-NTA resin, and elution by 50–500mM imidizaole gradient in binding buffer (20mM Hepes/KOH pH 7, 200mM KCl, 2mM β-mercaptoethanol, 10% glycerol). His6-NSF was then incubated with GST-Plk2c or GST-GluA2c (aa834–883), with ATP, ADP or ATP-γ-S (all at 2mM) in binding buffer. For ATPase assay, 1 μg His6-NSF was incubated in ATPase buffer (25mM Tris-HCl, pH 8.8, 100mM KCl, 0.5mM DTT, 2mM MgCl2, 0.6mM purified ATP, 10% glycerol) at 37°C with 10 μg of GST, GST-GluA2c, or GST-Plk2c. An ATPase colorimetric assay (Innova Biosciences) was used according to manufacturer's directions to detect formation of free phosphate and values read at 595nm.
Cell culture, Transfection, Sindbis virus infection and Picrotoxin induction
COS-7 cells were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum and 0.1% gentamicin (Invitrogen). COS-7 cells were transiently transfected with 1μg of plasmid DNA using Lipofectamine 2000 (Invitrogen), expressed for 24 h and harvested in IP buffer (see above). Primary hippocampal neurons were prepared from day 18 rat embryos and maintained 18–24 DIV. All animals were used in accordance with guidelines of the Georgetown University Animal Care and Use Committee. Cells were plated at medium density (~150 cells/mm2
) on coverslips coated with poly-D-lysine (Sigma) and laminin (2μg/mL, Roche). Cultures were grown in Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen), 0.5mM glutamine and 12.5 μM glutamate. Neurons were transfected using calcium phosphate as described14
or Lipofectamine 2000 (Invitrogen). Transfected neurons were incubated for 1–2 days, 3 days for RNAi experiments. For some experiments picrotoxin (100μM, Tocris Bioscience) was applied to neurons for 24 h. Plk2, K108M, or GFP were cloned into the pSinRep5 Sindbis virus vector (Invitrogen) and replication-defective pseudovirions produced according to the manufacturer's directions. Neurons were DIV18–21 at time of infection and duration of infection limited to 18–24 h, during which no significant toxicity could be observed.
Immunoprecipitation and immunoblotting
For immunoprecipitation, cells were lysed in IP buffer (see above), centrifuged to remove cell debris, and incubated ~18 h at 4°C with 1 μg antibody. Complexes were precipitated with Protein A-sepharose or glutathione sepharose, as appropriate. Bound proteins were eluted by boiling in Laemmli sample buffer, centrifuged and the supernatants separated by SDS-PAGE, transferred to nitrocellulose and imaged with enhanced chemiluminescence (Western Lightning (Perkin Elmer) or SuperSignal West Femto (Pierce)) following HRP-linked secondary antibody.
For immunolabeling of cell surface receptors, infected primary hippocampal cultured neurons DIV19–22 were treated with primary N-terminal specific antibody under nonpermeabilizing conditions, fixed, washed and treated with saturating concentrations of fluorescently-tagged secondary antibody. Cells were permeabilized and immunostained for Plk2 (or GFP) to visualize infected cells. Antibodies for immunostaining were diluted in conditioned media or ADB buffer (3% normal goat serum, 0.1% BSA, PBS) prior to permeabilization and GDB buffer (0.1% gelatin, 0.3% Triton X-100, 16mM sodium phosphate pH 7.4, 450mM NaCl), thereafter. For experiments with NEM, drug was applied for 10min at 10μM immediately before immunolabeling.
Cell Surface Biotinylation
COS-7 cells were transfected with GluA2 and combinations of NSF and Plk2 (WT or KN) and expressed for 24 h. Cells were washed twice with PBS, and surface proteins labeled with Sulfo-NHS-SS-Biotin (Pierce) (10 μl at 500 μg/ml in PBS) with gentle shaking at 4°C for 30 min. Cells were bathed in quenching solution (50μL, Pierce), washed twice with Tris-buffered saline, lysed in 500 μl of IP buffer, collected and disrupted by sonication on ice. After 30-min incubation on ice, cell lysates were clarified by centrifugation (10,000×g, 2 min). To isolate biotin-labeled proteins, lysates were added to immobilized NeutrAvidin TM Gel (50 μl) and incubated for 1 h at room temperature. Gels were washed 5 times with wash buffer and incubated 1 h with SDS-PAGE sample buffer including 50mM dithiothreitol. Surface proteins were then analyzed by immunoblotting for GluA2. For neurons, labeling was performed in ice-cold ACSF for 60 min, and lysis was performed in RIPA buffer. Intracellular receptor pool was calculated as [(total – surface)/total receptors].
Fluorescence Lifetime Microscopy
Proximity of NSF and Plk2 was assessed by FLIM, a fluorescence resonance energy transfer-based technique using multiphoton microscopy. Myc-tagged NSF and HA-tagged Plk2 were transfected into COS-7 cells and labeled by indirect immunocytochemistry using mouse myc and rabbit HA antibodies, and then labeled with secondary Alexa488 anti-mouse (donor fluorophore) and Cy3-anti-rabbit secondary as the acceptor fluorophore. Lifetime of the Alexa488 donor fluorophore attached to NSF decreases due to nonradiative energy transfer if Cy3-Plk2 (acceptor fluorophore) is positioned within 1–10 nm of the donor. Thus, a decreased donor lifetime indicates proximity of the two fluorophores (and the proteins labeled with these fluorophores). Images of Alexa488 fluorescence lifetimes on a pixel-by-pixel basis were acquired using a Bio-Rad Radiance 2000 multiphoton microscope, Ti:sapphire laser (Spectra Physics), a high-speed detector MCP R3809 (Hamamatsu), and a time-correlated single-photon counting acquisition board (SPC 830, Becker and Hickl GmbH). Images were analyzed using SPC-Image V2.8 (Becker and Hickl GmbH). Donor fluorophore lifetimes were fit using this software to two exponential decay curves to determine a fraction of the donor interacting with an acceptor. Results are presented in pseudocolored images representing color-coded donor lifetime on a pixel-by-pixel basis.
Internalization and recycling assay
Cultured hippocampal neurons were live labeled with 1° antibody recognizing GluA2 N-terminus. After incubation with antibody, cells were returned to conditioned media at 37°C, 5% CO2 for 0–30 min to allow receptor endocytosis. Cells were then fixed and remaining surface GluA2 labeled using saturating concentrations of 2° antibody. Following permeabilization, internalized receptors were labeled using a different 2° antibody. For recycling assays, the same procedure as above was followed except that cells were returned to conditioned media at 37°C, 5% CO2 for 30 min to allow for receptor internalization. Remaining surface-bound primary antibody was then acid stripped (0.5M NaCl and 0.2M acetic acid, at 4°C, 2 min). Neurons were incubated at 37°C, 5% CO2 for 1 hour with 2° antibody to allow for and label internalized receptors recycling back to the cell surface, or immediately fixed and incubated with 2° antibody to verify complete acid stripping. After washing, neurons were subsequently fixed and permeabilized and immunolabeled to show transfected cells (GFP or Plk2).
Yeast two-hybrid assay
Yeast two-hybrid analysis was conducted as described15
. NSF was cloned into the prey vector, pGAD, and fragments of Plk2c cloned into the bait vector, pBHA. L40 yeast strain and beta-galactosidase reporter were used to assay for interaction of NSF and Plk2 fragments.
Maltose binding protein fusion purification and pull-down
Plk2 PBind was cloned into the vector pMAL-c2× (New England Biolabs), expressed in E.coli, and purified according to the manufacturer's directions with amylose resin and maltose elution. Purified MBP-PBind or MBP alone (10 μg) was added to binding buffer (20mM Tris pH 7.4, 150mM NaCl) with 200 μg of whole brain homogenate (extracted with 1% deoxycholate, clarified by centrifugation at 100,000×g, and dialyzed against PBS) and 10 μl amylose beads for 2 h at 4°C with gentle agitation. Pellets were washed 3× with 20mM Tris pH 7.4, 500mM NaCl and analyzed by immunoblotting against NSF as well as Coomassie staining for fusion proteins.
Quantification and image analysis
Images were acquired using an Axiovert 200M epifluorescnece inverted microscope (Zeiss) using consistent laser intensity or camera exposure levels for each fluorescent marker in each experiment. For image analysis and quantification, measurements were made using MetaMorph software (Molecular Devices). Average intensity was calculated from integrated intensity and area for each selected area. Secondary dendritic branches (>20μm from the cell body) were used for quantification except where noted.
Surface plasmon resonance (SPR)
Biacore T100 instrument was used to measure direct interaction between recombinant NSF protein and synthetic Plk2 peptides, pep-PBind (sequence SAVENKQQIGDAIRMIVRGTLGSC) and a scrambled version, pep-scr (sequence SGDISVKINARITVCARLQGEMQG). Biacore series S NTA sensor chip was charged with 3 min injection of 100mM Ni2SO4. Injection of 122nM NSF protein for 4 min captured 5842 RU ligand on flow cell 2. Ni+ charged flow cell 1 was used as reference surface. Peptides were injected at 1 μM concentration for 1 min. Injections were repeated 3 times. All injections were made at 10μl/min flow rate. HBS-P (10mM Hepes pH 7.4, 150mM NaCl, 0.05% P-20) buffer supplemented with 2mM ATP was used as running and dilution buffers.
Wistar rats 14–18 days old were anesthetized with isoflurane and decapitated in accordance with NIH animal care and use guidelines. Transverse hippocampal slices (400 μm) were cut in ice-cold artificial cerebrospinal fluid (ACSF) containing (mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 9 MgSO4, 1 NaH2PO4, 26.2 NaHCO3, 11 glucose equilibrated with 95% O2 and 5% CO2. Slices were allowed to recover for at least 1 hour in ACSF at room temperature (composition as above except 1.3mM MgSO4). Whole cell patch clamp somatic recordings were made from visually identified CA1 pyramidal neurons in the presence of 50 μM picrotoxin at room temperature. The whole cell solution contained (mM): 135 CsMeSO4; 8 NaCl; 10 HEPES; 0.5 EGTA; 4 Mg-ATP; 0.3 Na-GTP; 5 QX-314 (pH 7.25), 285 mOsm. In addition, 100 μM of peptide was included together with 100 μM of each of the following protease inhibitors: bestatin, leupeptin, and pepstatin-A. EPSCs (at a holding potential of −70 mV) were evoked by afferent stimulation (0.1Hz) of fibers in the stratum radiatum. All recordings were made using a Multiclamp 700B amplifier (Axon Instruments), signals were filtered at 4 kHz, digitized at 10 kHz, and analyzed on-line using pClamp 9.2 (Axon Instruments). EPSCs were averaged during the first and last 2 minutes of recording for statistical analysis.
All values were expressed as means ± SEM, unless otherwise stated. Experiments were performed at least in triplicate and Student's t-test used for pairwise tests of significance, and ANOVA with Tukey's post-hoc test for group comparisons.