MORC2 phosphorylation fine tunes its DNA compaction activity.

Variants in the poorly characterised oncoprotein, MORC2, a chromatin remodelling ATPase, lead to defects in epigenetic regulation and DNA damage response. The C-terminal domain (CTD) of MORC2, frequently phosphorylated in DNA damage, promotes cancer progression, but its role in chromatin remodelling remains unclear. Here, we report a molecular characterisation of full-length, phosphorylated MORC2, demonstrating its preference for binding open chromatin and functioning as a DNA sliding clamp.

Co-clustering of EphB6 and ephrinB1 in trans restrains cancer cell invasion.

EphB6 is an understudied ephrin receptor tyrosine pseudokinase that is downregulated in multiple types of metastatic cancers. Unlike its kinase-active counterparts which autophosphorylate and transmit signals upon intercellular interaction, little is known about how EphB6 functions in the absence of intrinsic kinase activity. Here, we unveil a molecular mechanism of cell-cell interaction driven by EphB6.

Cryo-EM structure of the extracellular domain of murine Thrombopoietin Receptor in complex with Thrombopoietin.

Thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for haematopoetic stem cell maintenance. Tpo functions by binding its receptor (TpoR, a homodimeric Class I cytokine receptor) and initiating cell proliferation or differentiation. Here we characterise the murine Tpo:TpoR signalling complex biochemically and structurally, using cryo-electron microscopy.

Interaction of PINK1 with nucleotides and kinetin.

The ubiquitin kinase PINK1 accumulates on damaged mitochondria to trigger mitophagy, and PINK1 loss-of-function mutations cause early onset Parkinson's disease. Nucleotide analogs such as kinetin triphosphate (KTP) were reported to enhance PINK1 activity and may represent a therapeutic strategy for the treatment of Parkinson's disease. Here, we investigate the interaction of PINK1 with nucleotides, including KTP.

Structures of the interleukin 11 signalling complex reveal gp130 dynamics and the inhibitory mechanism of a cytokine variant.

Interleukin (IL-)11, an IL-6 family cytokine, has pivotal roles in autoimmune diseases, fibrotic complications, and solid cancers. Despite intense therapeutic targeting efforts, structural understanding of IL-11 signalling and mechanistic insights into current inhibitors are lacking. Here we present cryo-EM and crystal structures of the human IL-11 signalling complex, including the complex containing the complete extracellular domains of the shared IL-6 family β-receptor, gp130.

Phosphorylation-dependent pseudokinase domain dimerization drives full-length MLKL oligomerization.

The necroptosis pathway is a lytic, pro-inflammatory mode of cell death that is widely implicated in human disease, including renal, pulmonary, gut and skin inflammatory pathologies. The precise mechanism of the terminal steps in the pathway, where the RIPK3 kinase phosphorylates and triggers a conformation change and oligomerization of the terminal pathway effector, MLKL, are only emerging. Here, we structurally identify RIPK3-mediated phosphorylation of the human MLKL activation loop as a cue for MLKL pseudokinase domain dimerization.

Biparatopic nanobodies targeting the receptor binding domain efficiently neutralize SARS-CoV-2.

The development of therapeutics to prevent or treat COVID-19 remains an area of intense focus. Protein biologics, including monoclonal antibodies and nanobodies that neutralize virus, have potential for the treatment of active disease. Here, we have used yeast display of a synthetic nanobody library to isolate nanobodies that bind the receptor-binding domain (RBD) of SARS-CoV-2 and neutralize the virus.

Activation mechanism of PINK1.

Mutations in the protein kinase PINK1 lead to defects in mitophagy and cause autosomal recessive early onset Parkinson's disease. PINK1 has many unique features that enable it to phosphorylate ubiquitin and the ubiquitin-like domain of Parkin. Structural analysis of PINK1 from diverse insect species with and without ubiquitin provided snapshots of distinct structural states yet did not explain how PINK1 is activated.

Monoubiquitination by the human Fanconi anemia core complex clamps FANCI:FANCD2 on DNA in filamentous arrays.

FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by the Fanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here, we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination.

The structure of the PA28-20S proteasome complex from Plasmodium falciparum and implications for proteostasis.

The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress.

Structural Analysis of Polysaccharide Networks by Transmission Electron Microscopy: Comparison with Small-Angle X-ray Scattering.

Polysaccharide gels assembled from the anionic biopolymers pectin and carrageenan have been studied using transmission electron microscopy (TEM). Gels were formed in several different ways: for pectin, hydrogen bonding was used to form junction zones between strands, whereas for carrageenan systems, several different ion types were used to form ionotropic networks. Using this approach, several distinct network architectures were realized.

The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV.

The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time.

Nuclear Pore-Like Structures in a Compartmentalized Bacterium.

Planctomycetes are distinguished from other Bacteria by compartmentalization of cells via internal membranes, interpretation of which has been subject to recent debate regarding potential relations to Gram-negative cell structure. In our interpretation of the available data, the planctomycete Gemmata obscuriglobus contains a nuclear body compartment, and thus possesses a type of cell organization with parallels to the eukaryote nucleus. Here we show that pore-like structures occur in internal membranes of G.

Preliminary characterization of Tasmanian aquareovirus (TSRV) isolates.

In an attempt to determine whether or not genetic variants of the Tasmanian strain of Atlantic salmon aquareovirus (TSRV) exist, 14 isolates of TSRV, originating from various locations in Tasmania, covering a 20-year period (1990-2010), obtained from various host species and tissues, and isolated on different cell lines, were selected for this study. Two categories, termed "typical" and "atypical", of variants of TSRV were identified based on preliminary genotypic and phenotypic characterization carried out on these 14 different isolates. In addition, electron microscopic examination indicated the existence of at least three variants based on viral particle size.

Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry.

Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane.

Zooming in: Structural Investigations of Rheologically Characterized Hydrogen-Bonded Low-Methoxyl Pectin Networks.

Self-assembled hydrogen-bonded networks of the polysaccharide pectin, a mechanically functional component of plant cell walls, have been of recent interest as biomimetic exemplars of physical gels, and the microrheological and strain-stiffening behaviors have been previously investigated. Despite this detailed rheological characterization of preformed gels, little is known about the fundamental arrangement of the polymers into cross-linking junction zones, the size of these bonded regions, and the resultant network architecture in these hydrogen-bonded materials, especially in contrast to the plethora of such information available for their well-known calcium-assembled counterparts. In this work, in concert with pertinent rheological measurements, an in-depth structural study of the hydrogen-bond-mediated gelation of pectins is provided.

Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.

Novel formulation of a reconstituted high-density lipoprotein (CSL112) dramatically enhances ABCA1-dependent cholesterol efflux.

Objective: The ability of high-density lipoprotein (HDL) to remove cholesterol from atherosclerotic plaque is thought to underlie its inverse correlation with cardiovascular risk. Our objective was to produce and characterize a human apolipoprotein AI (apoA-I) product optimized to treat clinical atherosclerotic disease.

Approach And Results: A new formulation of full length, plasma-derived human apoA-I termed CSL112 was designed to maximize the cholesterol efflux from cells and exhibit favorable pharmacological properties.

Visualising single molecules of HIV-1 and miRNA nucleic acids.

Background: The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19-26 nucleotides in length.

Results: We used O-FISH to visualize miR146a in human and avian cells.

Micromachining tools and correlative approaches for cellular cryo-electron tomography.

A principal limitation of cryo-transmission electron microscopy performed on cells or tissues is the accessible specimen thickness. This is exacerbated in tomography applications, where the aspect ratio (and thus the apparent specimen thickness) changes considerably during specimen tilting. Cryo-ultramicrotomy is the most obvious way of dealing with this problem; however, frozen-hydrated sections suffer from potentially inconsistent compression that cannot be corrected with certainty, and furthermore, yields of sections that satisfy all of the conditions necessary for tomographic imaging are poor.

Correlative cryo-light microscopy and cryo-electron tomography: from cellular territories to molecular landscapes.

In cell biology, visual techniques such as light and electron microscopy provide the most intuitive means by which to study structure and function; however, no single microscopy technique is capable of providing all of the desired information. As a consequence, many separate techniques have evolved, each with unique capabilities. The most informative approaches are global in the sense that they take advantage of multiple imaging modalities spanning a range of spatial scales and frequencies, preferably encompassing preservation of the hydrated nature of the cell.

Visualizing cells at the nanoscale.

Cryogenic electron tomography (cryo- ET) enables the 3D visualization of biological material at a previously unseeable scale. Carefully controlled cryogenic specimen preparation avoids the artefacts that are notorious to conventional electron microscopy specimen preparation. To date, studies employing cryo- ET have mostly been restricted to isolated macromolecular assemblies, small prokaryotic cells or thin regions of eukaryotic cells owing to the limited penetration depth of electrons through ice-embedded preparations.

Chromatin organization and radio resistance in the bacterium Gemmata obscuriglobus.

The organization of chromatin has a major impact on cellular activities, such as gene expression. For bacteria, it was suggested that the spatial organization of the genetic material correlates with transcriptional levels, implying a specific architecture of the chromosome within the cytoplasm. Accordingly, recent technological advances have emphasized the organization of the genetic material within nucleoid structures.