TY - CHAP
AB - Here we describe the in vivo DNA assembly approach, where molecular cloning procedures are performed using an E. coli recA-independent recombination pathway, which assembles linear fragments of DNA with short homologous termini. This pathway is present in all standard laboratory E. coli strains and, by bypassing the need for in vitro DNA assembly, allows simplified molecular cloning to be performed without the plasmid instability issues associated with specialized recombination-cloning bacterial strains. The methodology requires specific primer design and can perform all standard plasmid modifications (insertions, deletions, mutagenesis, and sub-cloning) in a rapid, simple, and cost-efficient manner, as it does not require commercial kits or specialized bacterial strains. Additionally, this approach can be used to perform complex procedures such as multiple modifications to a plasmid, as up to 6 linear fragments can be assembled in vivo by this recombination pathway. Procedures generally require less than 3 h, involving PCR amplification, DpnI digestion of template DNA, and transformation, upon which circular plasmids are assembled. In this chapter we describe the requirements, procedure, and potential pitfalls when using this technique, as well as protocol variations to overcome the most common issues.
AU - Arroyo-Urea, Sandra
AU - Watson, Jake
AU - García-Nafría, Javier
ED - Scarlett, Garry
ID - 12720
SN - 1064-3745
T2 - DNA Manipulation and Analysis
TI - Molecular Cloning Using In Vivo DNA Assembly
VL - 2633
ER -
TY - JOUR
AB - AMPA glutamate receptors (AMPARs) mediate excitatory neurotransmission throughout the brain. Their signalling is uniquely diversified by brain region-specific auxiliary subunits, providing an opportunity for the development of selective therapeutics. AMPARs associated with TARP γ8 are enriched in the hippocampus, and are targets of emerging anti-epileptic drugs. To understand their therapeutic activity, we determined cryo-EM structures of the GluA1/2-γ8 receptor associated with three potent, chemically diverse ligands. We find that despite sharing a lipid-exposed and water-accessible binding pocket, drug action is differentially affected by binding-site mutants. Together with patch-clamp recordings and MD simulations we also demonstrate that ligand-triggered reorganisation of the AMPAR-TARP interface contributes to modulation. Unexpectedly, one ligand (JNJ-61432059) acts bifunctionally, negatively affecting GluA1 but exerting positive modulatory action on GluA2-containing AMPARs, in a TARP stoichiometry-dependent manner. These results further illuminate the action of TARPs, demonstrate the sensitive balance between positive and negative modulatory action, and provide a mechanistic platform for development of both positive and negative selective AMPAR modulators.
AU - Zhang, Danyang
AU - Lape, Remigijus
AU - Shaikh, Saher A.
AU - Kohegyi, Bianka K.
AU - Watson, Jake
AU - Cais, Ondrej
AU - Nakagawa, Terunaga
AU - Greger, Ingo H.
ID - 12786
JF - Nature Communications
TI - Modulatory mechanisms of TARP γ8-selective AMPA receptor therapeutics
VL - 14
ER -
TY - JOUR
AB - Three-dimensional (3D) reconstruction of living brain tissue down to an individual synapse level would create opportunities for decoding the dynamics and structure–function relationships of the brain’s complex and dense information processing network; however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise ratio and prohibitive light burden in optical imaging, whereas electron microscopy is inherently static. Here we solved these challenges by developing an integrated optical/machine-learning technology, LIONESS (live information-optimized nanoscopy enabling saturated segmentation). This leverages optical modifications to stimulated emission depletion microscopy in comprehensively, extracellularly labeled tissue and previous information on sample structure via machine learning to simultaneously achieve isotropic super-resolution, high signal-to-noise ratio and compatibility with living tissue. This allows dense deep-learning-based instance segmentation and 3D reconstruction at a synapse level, incorporating molecular, activity and morphodynamic information. LIONESS opens up avenues for studying the dynamic functional (nano-)architecture of living brain tissue.
AU - Velicky, Philipp
AU - Miguel Villalba, Eder
AU - Michalska, Julia M
AU - Lyudchik, Julia
AU - Wei, Donglai
AU - Lin, Zudi
AU - Watson, Jake
AU - Troidl, Jakob
AU - Beyer, Johanna
AU - Ben Simon, Yoav
AU - Sommer, Christoph M
AU - Jahr, Wiebke
AU - Cenameri, Alban
AU - Broichhagen, Johannes
AU - Grant, Seth G.N.
AU - Jonas, Peter M
AU - Novarino, Gaia
AU - Pfister, Hanspeter
AU - Bickel, Bernd
AU - Danzl, Johann G
ID - 13267
JF - Nature Methods
SN - 1548-7091
TI - Dense 4D nanoscale reconstruction of living brain tissue
VL - 20
ER -
TY - JOUR
AB - Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanometer synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS uses fixation-compatible extracellular labeling and optical imaging, including stimulated emission depletion or expansion microscopy, to comprehensively delineate cellular structures. It enables three-dimensional reconstruction of single synapses and mapping of synaptic connectivity by identification and analysis of putative synaptic cleft regions. Applying CATS to the mouse hippocampal mossy fiber circuitry, we reconstructed and quantified the synaptic input and output structure of identified neurons. We furthermore demonstrate applicability to clinically derived human tissue samples, including formalin-fixed paraffin-embedded routine diagnostic specimens, for visualizing the cellular architecture of brain tissue in health and disease.
AU - Michalska, Julia M
AU - Lyudchik, Julia
AU - Velicky, Philipp
AU - Korinkova, Hana
AU - Watson, Jake
AU - Cenameri, Alban
AU - Sommer, Christoph M
AU - Amberg, Nicole
AU - Venturino, Alessandro
AU - Roessler, Karl
AU - Czech, Thomas
AU - Höftberger, Romana
AU - Siegert, Sandra
AU - Novarino, Gaia
AU - Jonas, Peter M
AU - Danzl, Johann G
ID - 14257
JF - Nature Biotechnology
SN - 1087-0156
TI - Imaging brain tissue architecture across millimeter to nanometer scales
ER -
TY - JOUR
AB - AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory
synapses in the brain. Glutamate binding to the receptor’s ligand-binding domains (LBDs)
leads to ion channel activation and desensitization. Gating kinetics shape synaptic transmission
and are strongly modulated by transmembrane AMPAR regulatory proteins (TARPs)
through currently incompletely resolved mechanisms. Here, electron cryo-microscopy
structures of the GluA1/2 TARP-γ8 complex, in both open and desensitized states
(at 3.5 Å), reveal state-selective engagement of the LBDs by the large TARP-γ8 loop (‘β1’),
elucidating how this TARP stabilizes specific gating states. We further show how TARPs alter
channel rectification, by interacting with the pore helix of the selectivity filter. Lastly, we
reveal that the Q/R-editing site couples the channel constriction at the filter entrance to the
gate, and forms the major cation binding site in the conduction path. Our results provide a
mechanistic framework of how TARPs modulate AMPAR gating and conductance.
AU - Herguedas, Beatriz
AU - Kohegyi, Bianka K.
AU - Dohrke, Jan Niklas
AU - Watson, Jake
AU - Zhang, Danyang
AU - Ho, Hinze
AU - Shaikh, Saher A.
AU - Lape, Remigijus
AU - Krieger, James M.
AU - Greger, Ingo H.
ID - 10763
JF - Nature Communications
TI - Mechanisms underlying TARP modulation of the GluA1/2-γ8 AMPA receptor
VL - 13
ER -
TY - GEN
AB - Complex wiring between neurons underlies the information-processing network enabling all brain functions, including cognition and memory. For understanding how the network is structured, processes information, and changes over time, comprehensive visualization of the architecture of living brain tissue with its cellular and molecular components would open up major opportunities. However, electron microscopy (EM) provides nanometre-scale resolution required for full in-silico reconstruction1–5, yet is limited to fixed specimens and static representations. Light microscopy allows live observation, with super-resolution approaches6–12 facilitating nanoscale visualization, but comprehensive 3D-reconstruction of living brain tissue has been hindered by tissue photo-burden, photobleaching, insufficient 3D-resolution, and inadequate signal-to-noise ratio (SNR). Here we demonstrate saturated reconstruction of living brain tissue. We developed an integrated imaging and analysis technology, adapting stimulated emission depletion (STED) microscopy6,13 in extracellularly labelled tissue14 for high SNR and near-isotropic resolution. Centrally, a two-stage deep-learning approach leveraged previously obtained information on sample structure to drastically reduce photo-burden and enable automated volumetric reconstruction down to single synapse level. Live reconstruction provides unbiased analysis of tissue architecture across time in relation to functional activity and targeted activation, and contextual understanding of molecular labelling. This adoptable technology will facilitate novel insights into the dynamic functional architecture of living brain tissue.
AU - Velicky, Philipp
AU - Miguel Villalba, Eder
AU - Michalska, Julia M
AU - Wei, Donglai
AU - Lin, Zudi
AU - Watson, Jake
AU - Troidl, Jakob
AU - Beyer, Johanna
AU - Ben Simon, Yoav
AU - Sommer, Christoph M
AU - Jahr, Wiebke
AU - Cenameri, Alban
AU - Broichhagen, Johannes
AU - Grant, Seth G. N.
AU - Jonas, Peter M
AU - Novarino, Gaia
AU - Pfister, Hanspeter
AU - Bickel, Bernd
AU - Danzl, Johann G
ID - 11943
T2 - bioRxiv
TI - Saturated reconstruction of living brain tissue
ER -
TY - GEN
AB - Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanoscopic synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS leverages fixation-compatible extracellular labeling and advanced optical readout, in particular stimulated-emission depletion and expansion microscopy, to comprehensively delineate cellular structures. It enables 3D-reconstructing single synapses and mapping synaptic connectivity by identification and tailored analysis of putative synaptic cleft regions. Applying CATS to the hippocampal mossy fiber circuitry, we demonstrate its power to reveal the system’s molecularly informed ultrastructure across spatial scales and assess local connectivity by reconstructing and quantifying the synaptic input and output structure of identified neurons.
AU - Michalska, Julia M
AU - Lyudchik, Julia
AU - Velicky, Philipp
AU - Korinkova, Hana
AU - Watson, Jake
AU - Cenameri, Alban
AU - Sommer, Christoph M
AU - Venturino, Alessandro
AU - Roessler, Karl
AU - Czech, Thomas
AU - Siegert, Sandra
AU - Novarino, Gaia
AU - Jonas, Peter M
AU - Danzl, Johann G
ID - 11950
T2 - bioRxiv
TI - Uncovering brain tissue architecture across scales with super-resolution light microscopy
ER -
TY - JOUR
AB - AMPA receptors (AMPARs) mediate the majority of excitatory transmission in the brain and enable the synaptic plasticity that underlies learning1. A diverse array of AMPAR signalling complexes are established by receptor auxiliary subunits, which associate with the AMPAR in various combinations to modulate trafficking, gating and synaptic strength2. However, their mechanisms of action are poorly understood. Here we determine cryo-electron microscopy structures of the heteromeric GluA1–GluA2 receptor assembled with both TARP-γ8 and CNIH2, the predominant AMPAR complex in the forebrain, in both resting and active states. Two TARP-γ8 and two CNIH2 subunits insert at distinct sites beneath the ligand-binding domains of the receptor, with site-specific lipids shaping each interaction and affecting the gating regulation of the AMPARs. Activation of the receptor leads to asymmetry between GluA1 and GluA2 along the ion conduction path and an outward expansion of the channel triggers counter-rotations of both auxiliary subunit pairs, promoting the active-state conformation. In addition, both TARP-γ8 and CNIH2 pivot towards the pore exit upon activation, extending their reach for cytoplasmic receptor elements. CNIH2 achieves this through its uniquely extended M2 helix, which has transformed this endoplasmic reticulum-export factor into a powerful AMPAR modulator that is capable of providing hippocampal pyramidal neurons with their integrative synaptic properties.
AU - Zhang, Danyang
AU - Watson, Jake
AU - Matthews, Peter M.
AU - Cais, Ondrej
AU - Greger, Ingo H.
ID - 9549
JF - Nature
SN - 0028-0836
TI - Gating and modulation of a hetero-octameric AMPA glutamate receptor
VL - 594
ER -
TY - JOUR
AB - AMPA receptor (AMPAR) abundance and positioning at excitatory synapses regulates the strength of transmission. Changes in AMPAR localisation can enact synaptic plasticity, allowing long-term information storage, and is therefore tightly controlled. Multiple mechanisms regulating AMPAR synaptic anchoring have been described, but with limited coherence or comparison between reports, our understanding of this process is unclear. Here, combining synaptic recordings from mouse hippocampal slices and super-resolution imaging in dissociated cultures, we compare the contributions of three AMPAR interaction domains controlling transmission at hippocampal CA1 synapses. We show that the AMPAR C-termini play only a modulatory role, whereas the extracellular N-terminal domain (NTD) and PDZ interactions of the auxiliary subunit TARP γ8 are both crucial, and each is sufficient to maintain transmission. Our data support a model in which γ8 accumulates AMPARs at the postsynaptic density, where the NTD further tunes their positioning. This interplay between cytosolic (TARP γ8) and synaptic cleft (NTD) interactions provides versatility to regulate synaptic transmission and plasticity.
AU - Watson, Jake
AU - Pinggera, Alexandra
AU - Ho, Hinze
AU - Greger, Ingo H.
ID - 9985
IS - 1
JF - Nature Communications
TI - AMPA receptor anchoring at CA1 synapses is determined by N-terminal domain and TARP γ8 interactions
VL - 12
ER -