Malinow: Difference between revisions

From bradwiki

Jump to navigation Jump to search

Revision as of 22:32, 12 July 2013

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 (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)

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)

Experiments

xxxAUTHORSsxxx • xxxYEARxxx • xxxJOURNALxxx - PDF

Expand to view experiment summary

Abstract

abstract

xxxAUTHORSsxxx • xxxYEARxxx • xxxJOURNALxxx - PDF

Expand to view experiment summary

Abstract

abstract

xxxAUTHORSsxxx • xxxYEARxxx • xxxJOURNALxxx - PDF

Expand to view experiment summary

Abstract

abstract

xxxAUTHORSsxxx • xxxYEARxxx • xxxJOURNALxxx - PDF

Expand to view experiment summary

Abstract

abstract

xxxAUTHORSsxxx • xxxYEARxxx • xxxJOURNALxxx - PDF

Expand to view experiment summary

Abstract

abstract

xxxAUTHORSsxxx • xxxYEARxxx • xxxJOURNALxxx - PDF

Expand to view experiment summary

Abstract

abstract

RANDOM NOTES

Proteins that interact with AMPARs

Error creating thumbnail: File missing

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

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

Retrieved from "https://bradleymonk.com/wiki/index.php?title=Malinow&oldid=1421"