Malinow

Malinow

Molecular Methods

Quantum Dots

Choquet

AMPAR

Experiment Ideas

experimental notes and highlighted findings

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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 (Lu Ting 2013 PLOSONE)
• AMPAR-peptide A, QD-peptide B, which binds peptide A (Zhang Kodadek 2000 NatBiotech)
• AMPAR-unnatural amino acid azide, QD-propargyl (Chaterjee Schultz 2013 PNAS)

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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)

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Experiments

Hayashi, Shi, Esteban, Piccini, Poncer, Malinow • 2000 • Science - PDF

Driving AMPA Receptors into Synapses by LTP and CaMKII: Requirement for GluR1 and PDZ Domain Interaction

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Abstract

• To elucidate mechanisms that control and execute activity-dependent synaptic plasticity, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA-Rs) with an electrophysiological tag were expressed in rat hippocampal neurons. Long-term potentiation (LTP) or increased activity of the calcium/calmodulin-dependent protein kinase II (CaMKII) induced delivery of tagged AMPA-Rs into synapses. This effect was not diminished by mutating the CaMKII phosphorylation site on the GluR1 AMPA-R subunit, but was blocked by mutating a predicted PDZ domain interaction site. These results show that LTP and CaMKII activity drive AMPA-Rs to synapses by a mechanism that requires the association between GluR1 and a PDZ domain protein.

Kessels, Kopec, Klein, Malinow • 2009 • Nat Neurosci. - PDF

Roles of stargazin and phosphorylation in the control of AMPA receptor subcellular distribution

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Abstract

• Understanding how the subcellular fate of newly synthesized AMPA receptors (AMPARs) is controlled is important for elucidating the mechanisms of neuronal function. We examined the effect of increased synthesis of AMPAR subunits on their subcellular distribution in rat hippocampal neurons. Virally expressed AMPAR subunits (GluR1 or GluR2) accumulated in cell bodies and replaced endogenous dendritic AMPAR with little effect on total dendritic amounts and caused no change in synaptic transmission. Coexpressing stargazin (STG) or mimicking GluR1 phosphorylation enhanced dendritic GluR1 levels by protecting GluR1 from lysosomal degradation. However, STG interaction or GluR1 phosphorylation did not increase surface or synaptic GluR1 levels. Unlike GluR1, STG did not protect GluR2 from lysosomal degradation or increase dendritic GluR2 levels. In general, AMPAR surface levels, and not intracellular amounts, correlated strongly with synaptic levels. Our results suggest that AMPAR surface expression, but not its intracellular production or accumulation, is critical for regulating synaptic transmission.

Kopec, Real, Kessels, Malinow • 2007 • J Neuro - PDF

GluR1 links structural and functional plasticity at excitatory synapses

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Abstract

• Long-term potentiation (LTP), a cellular model of learning and memory, produces both an enhancement of synaptic function and an increase in the size of the associated dendritic spine. Synaptic insertion of AMPA receptors is known to play an important role in mediating the increase in synaptic strength during LTP, whereas the role of AMPA receptor trafficking in structural changes remains unexplored. Here, we examine how the cell maintains the correlation between spine size and synapse strength during LTP. We found that cells exploit an elegant solution by linking both processes to a single molecule: the AMPA-type glutamate receptor subunit 1 (GluR1). Synaptic insertion of GluR1 is required to permit a stable increase in spine size, both in hippocampal slice cultures and in vivo. Synaptic insertion of GluR1 is not sufficient to drive structural plasticity. Although crucial to the expression of LTP, the ion channel function of GluR1 is not required for the LTP-driven spine size enhancement. Remarkably, a recombinant cytosolic C-terminal fragment (C-tail) of GluR1 is driven to the postsynaptic density after an LTP stimulus, and the synaptic incorporation of this isolated GluR1 C-tail is sufficient to permit spine enlargement even when postsynaptic exocytosis of endogenous GluR1 is blocked. We conclude that during plasticity, synaptic insertion of GluR1 has two functions: the established role of increasing synaptic strength via its ligand-gated ion channel, and a novel role through the structurally stabilizing effect of its C terminus that permits an increase in spine size.

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RANDOM NOTES

Proteins that interact with AMPARs

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Song and Huganir 2002. Use of yeast two-hybrid method to identify AMPA-receptor-interacting proteins was crucial for the rapid progress in this field and has helped to identify a large complex of such proteins. AMPA-receptor-associated protein complex. AMPA receptors are associated with a large protein network. The GluR2 subunit specifically binds to several proteins, including glutamatereceptor- interacting protein (GRIP) 1, GRIP2, protein interacting with C kinase (PICK1) and N-ethylmaleimide sensitive factor (NSF). GRIP1, GRIP2 and PICK1 in turn bind to other proteins, including GRIP-associated proteins (GRASPs), EphB receptor tyrosine kinases, ephrins, kinesin 5 (KIF5) and protein kinase Cα (PKCα). The GluR1 subunit binds to synapse-associated protein 97 (SAP97, also known as hDLG) and protein 4.1 (4.1 N). All four AMPA-receptor subunits bind to neuronal activity regulated pentraxin (NARP) and stargazin. Stargazin binds, in turn, to the synaptic scaffolding protein postsynaptic density 95 (PSD95). These associated proteins appear to play an important role in the membrane trafficking of the receptors by escorting the receptor from the cell body to the synapse. In addition, this large complex might regulate novel downstream signal transduction pathways that emanate from the AMPA receptor. Abbreviations: CC, coiled-coil domain; GK, guanylate kinase domain; PDZ, PSD95/Dlg/ZO1 domain; SH3, SRC homology 3 domain.

Qdots

• Quantum Dots

• Qdot 655 Goat F(ab')2 Anti-Rabbit IgG Conjugate
• Qdot Methods Handbook
• Qdot Color Visualizer

Getting a Qdot into the cell

1. Conjugate Qdot with secondary antibody fab
2. Incubate tissue with primary antibodies for AMPAR and PSD95
3. Puff Qdots onto cell body, these will bind the primary at AMPAR N-terminus
4. When AMPARs internalize the Qdot will be dragged into cell
5. Cleave N-terminus of AMPAR to liberate Qdot
6. 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