Malinow
Malinow | Molecular Methods | Quantum Dots | Choquet | AMPAR |
Experiment Ideas
experimental notes and highlighted findings
Zac Email
Here are the different labeling techniques that might be applicable with recombinant expression of AMPARs. Roughly ranked from most to least likely to succeed, separated by large vs small AMPAR N-terminal additions. The references in parentheses are for background on the technique.
- Large AMPAR N-terminal addition
- Halotag (Promega): AMPAR-enzyme, QD-Halotag substrate
- AMPAR-streptavidin, QD-biotin
- Small AMPAR N-terminal addition
- AMPAR-FLAG, QD-anti-FLAG
- AMPAR-biotin ligase recognition peptide, QD-biotin + biotin ligase (LuTing2013PLOSONE)
- AMPAR-peptide A, QD-peptide B, which binds peptide A (ZhangKodadek2000NatBiotech)
- AMPAR-unnatural amino acid azide, QD-propargyl (ChaterjeeSchultz2013PNAS)
Choquet 2010 CaMKII triggers the diffusional trapping of surface AMPARs through phosphorylation of stargazin
- NMDAR activation promotes rapid translocation of aCaMKII::GFP to synapses, causing AMPAR trapping at 1 min (only synapses with CaMKII translocation)
- tCaMKII (active prion) promotes immobilization of endogenous GluR1 (containing) AMPARs (both synaptic and extrasynaptic), and to a much lesser extent GluA2 (containing) AMPARs.
- CaMKII direct phosphorylation of AMPARs unnecessary for synaptic trapping
- GluA1 - SAP97 interaction unnecessary for CaMKII-dependent synaptic trapping
- Stargazin increased tCaMKII-mediated trapping of recombinant GluA1 (homomeric), but tCaMKII had no effect on mobility of recombinant GluA2 (homomeric)
- Stargazin phosphorylation (by tCaMKII) is necessary for GluA1 trapping; blocking phosphorylation caused AMPAR mobility to significantly increase.
- intriguing finding: GluA1 subunit-specific effect of CaMKII, where it immobilizes recombinant GluA1 but not GluA2 homomeric AMPARs.
- findings consistent with specific role of GluA1 in activity-dependent trafficking - but Stargazin can bind all subunits??
- findings raise possibility that during LTP, CaMKII activation triggers both classical LTP and PPD. Interesting that LTP is frequently accompanied by PPD (opposite of PPF: paired-pulse facilitation)
RANDOM NOTES
Proteins that interact with AMPARs
Qdots
Getting a Qdot into the cell
- Conjugate Qdot with secondary antibody fab
- Incubate tissue with primary antibodies for AMPAR and PSD95
- Puff Qdots onto cell body, these will bind the primary at AMPAR N-terminus
- When AMPARs internalize the Qdot will be dragged into cell
- Cleave N-terminus of AMPAR to liberate Qdot
- Qdot can then bind the primary ligated to PSD95
Notes
- Molecular Methods
- FLASH technology
- Bredt
- minisog - gfp
- Acidic basic polypeptide recognition sequences
- Talk with nanotech group about various ways to conj. Qdots
- Nichol and England - couple Qdot to AMPAR agonist
- Have simulation be a competitive model where AMPARs are competing during LTP
- Quantitative review on synaptic numbers (Sheng)
PALM STORM
There are two major groups of methods for functional super-resolution microscopy:
1. Deterministic super-resolution: The most commonly used emitters in biological microscopy, fluorophores, show a nonlinear response to excitation, and this nonlinear response can be exploited to enhance resolution. These methods include STED, GSD, RESOLFT and SSIM.
2. Stochastical super-resolution PALM STORM: The chemical complexity of many molecular light sources gives them a complex temporal behaviour, which can be used to make several close-by fluorophores emit light at separate times and thereby become resolvable in time. These methods include SOFI and all single-molecule localization methods (SMLM) such as SPDM, SPDMphymod, PALM, FPALM, STORM and dSTORM.
NRSA
- Dominant negative PSD95