Neuropathic MORC2 mutations perturb GHKL ATPase dimerization dynamics and epigenetic silencing by multiple structural mechanisms

Missense mutations in MORC2 cause neuropathies including spinal muscular atrophy and Charcot-Marie-Tooth disease. We recently identified MORC2 as an effector of epigenetic silencing by the human silencing hub (HUSH). Here we report the biochemical and cellular activities of MORC2 variants, alongside crystal structures of wild-type and neuropathic forms of a human MORC2 fragment comprising the GHKL-type ATPase module and CW-type zinc finger. This fragment dimerizes upon binding ATP and contains a hinged, functionally critical coiled coil insertion absent in other GHKL ATPases. We find that dimerization and DNA binding of the MORC2 ATPase module transduce HUSH-dependent silencing. Disease mutations change the dynamics of dimerization by distinct structural mechanisms: destabilizing the ATPase-CW module, trapping the ATP lid or perturbing the dimer interface. These defects lead to modulation of HUSH function, thus providing a molecular basis for understanding MORC2-associated neuropathies

Cryo-electron tomography of NLRP3-activated ASC complexes reveals organelle co-localization.

NLRP3 induces caspase-1-dependent pyroptotic cell death to drive inflammation. Aberrant activity of NLRP3 occurs in many human diseases. NLRP3 activation induces ASC polymerization into a single, micron-scale perinuclear punctum. Higher resolution imaging of this signaling platform is needed to understand how it induces pyroptosis. Here, we apply correlative cryo-light microscopy and cryo-electron tomography to visualize ASC/caspase-1 in NLRP3-activated cells. The puncta are composed of branched ASC filaments, with a tubular core formed by the pyrin domain. Ribosomes and Golgi-like or endosomal vesicles permeate the filament network, consistent with roles for these organelles in NLRP3 activation. Mitochondria are not associated with ASC but have outer-membrane discontinuities the same size as gasdermin D pores, consistent with our data showing gasdermin D associates with mitochondria and contributes to mitochondrial depolarization

Visualisation of innate immune signalling complexes in cells by cryo-electron tomography

Innate immune signalling is a vital immune response to various infections and cell damage. A common feature of innate signalling complexes is forming a signalling platform through oligomerisation in cells. RIG-I-like receptors (RLRs), RIG-I, MDA5 and LGP2 sense cytosolic viral dsRNA and then promote oligomerisation of the mitochondrial antiviral- signalling protein (MAVS) on the outer mitochondrial membrane. MAVS signalling induces activation of cytosolic kinases, which in turn activates transcription factors IRF3 and NF-kB to promote interferon production. Similarly, activation of NLRP3, a sensor protein to various danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), induces oligomerisation of adaptor protein ASC into large puncta. Caspase-1 is recruited and then cleaved to initiate caspase-1 dependent pyroptosis and the release of pro-inflammatory cytokines. Previous structural studies have shown mechanistic models of the signalling components involved. However, the structure of the innate signalling complex has not been studied in cells so far. In this thesis, I tested fluorescence labelling strategies and employed live-cell confocal imaging techniques to visualise MAVS proteins in cells. In addition, I used cellular signalling assays to test the functionality of tagged-MAVS. Although the results has not yielded the desired functional and fluorescently labelled MAVS yet, it provides solid foundation for future labelling experiments of MAVS. I optimised a cryo-correlative light electron microscopy (cryo-CLEM) workflow to attain high-resolution structural insights into the ASC signalling complexes in cells. My cryo-ET reconstructions, obtained in unstained and fully hydrated conditions, show that the speck is formed of a filamentous network consisting of hollow-tube branched filaments with the dimensions predicted for ASC filaments, based on structural studies of purified ASC PYD filaments and full-length monomeric ASC. The structural organisation of the ASC filament network allows ribosomes and small TGN-like vesicles to be retained in the puncta, or to permeate through them. The filament branching and packing density within ASC puncta provide structural integrity while allowing downstream signalling molecules to diffuse freely and bind at high density within the network.Wellcome Trust Cambridge Trus

A bioactive phlebovirus-like envelope protein in a hookworm endogenous virus

Endogenous viral elements (EVEs), accounting for 15% of our genome, serve as a genetic reservoir from which new genes can emerge. Nematode EVEs are particularly diverse and informative of virus evolution. We identify Atlas virus – an intact retrovirus-like EVE in the human hookworm Ancylostoma ceylanicum, with an envelope protein genetically related to GN-GC glycoproteins from the Phenuiviridae family. A cryo-EM structure of Atlas GC reveals a class II viral membrane fusion protein fold not previously seen in retroviruses. Atlas GC has the structural hallmarks of an active fusogen. Atlas GC trimers insert into membranes with endosomal lipid compositions and low pH. When expressed on the plasma membrane, Atlas GC has cell-cell fusion activity. With its preserved biological activities, Atlas GC has the potential to acquire a cellular function. Our work reveals structural plasticity in reverse-transcribing RNA viruses

Neuropathic mutations in MORC2 perturb GHKL ATPase dimerization dynamics and epigenetic silencing by multiple structural mechanisms

Missense mutations in MORC2 cause neuropathies including spinal muscular atrophy and Charcot-Marie-Tooth disease. We recently identified MORC2 as an effector of epigenetic silencing by the HUSH complex. Here we report the biochemical and cellular activities of MORC2 variants, alongside crystal structures of wild-type and neuropathic forms of a human MORC2 fragment comprising the GHKL-type ATPase module and CW-type zinc finger. This fragment dimerizes upon binding ATP and contains a hinged, functionally critical coiled coil insertion absent in other GHKL ATPases. We find that dimerization and DNA binding of the MORC2 ATPase module transduce HUSH-dependent silencing. Disease mutations change the dynamics of dimerization by distinct structural mechanisms: destabilizing the ATPase-CW module, trapping the ATP lid or perturbing the dimer interface. These defects lead to modulation of HUSH function, thus providing a molecular basis for understanding MORC2-associated neuropathies.Wellcome Trust (101908/Z/13/Z and 101835/Z/13/Z) BBSRC (BB/N011791/1

Cryo-electron tomography of NLRP3-activated ASC complexes reveals organelle co-localization

AbstractNLRP3 induces caspase-1-dependent pyroptotic cell death to drive inflammation. Aberrant activity of NLRP3 occurs in many human diseases. NLRP3 activation induces ASC polymerization into a single, micron-scale perinuclear punctum. Higher resolution imaging of this signaling platform is needed to understand how it induces pyroptosis. Here, we apply correlative cryo-light microscopy and cryo-electron tomography to visualize ASC/caspase-1 in NLRP3-activated cells. The puncta are composed of branched ASC filaments, with a tubular core formed by the pyrin domain. Ribosomes and Golgi-like or endosomal vesicles permeate the filament network, consistent with roles for these organelles in NLRP3 activation. Mitochondria are not associated with ASC but have outer-membrane discontinuities the same size as gasdermin D pores, consistent with our data showing gasdermin D associates with mitochondria and contributes to mitochondrial depolarization.This work was supported by Senior Research Fellowships 101908/Z/13/Z and 217191/Z/19/Z from the Wellcome Trust to Y.M.; a PhD studentship from the China Scholarship Council and Cambridge Trust to Y.L.; and Investigator Award 108045/Z/15/Z from the Wellcome Trust to C.E.B

Cryo-electron tomography of NLRP3-activated ASC complexes reveals organelle co-localization.

Funder: China Scholarship Council (CSC); doi: https://doi.org/10.13039/501100004543NLRP3 induces caspase-1-dependent pyroptotic cell death to drive inflammation. Aberrant activity of NLRP3 occurs in many human diseases. NLRP3 activation induces ASC polymerization into a single, micron-scale perinuclear punctum. Higher resolution imaging of this signaling platform is needed to understand how it induces pyroptosis. Here, we apply correlative cryo-light microscopy and cryo-electron tomography to visualize ASC/caspase-1 in NLRP3-activated cells. The puncta are composed of branched ASC filaments, with a tubular core formed by the pyrin domain. Ribosomes and Golgi-like or endosomal vesicles permeate the filament network, consistent with roles for these organelles in NLRP3 activation. Mitochondria are not associated with ASC but have outer-membrane discontinuities the same size as gasdermin D pores, consistent with our data showing gasdermin D associates with mitochondria and contributes to mitochondrial depolarization.This work was supported by Senior Research Fellowships 101908/Z/13/Z and 217191/Z/19/Z from the Wellcome Trust to Y.M.; a PhD studentship from the China Scholarship Council and Cambridge Trust to Y.L.; and Investigator Award 108045/Z/15/Z from the Wellcome Trust to C.E.B