Researchers at the Crick are tackling the big questions about human health and disease, and new findings are published every week. Our faculty have picked some of the most significant papers published by Crick scientists, all of which are freely available thanks to our open science policy.
CD1d-mediated lipid presentation by CD11c+ cells regulates intestinal homeostasis
Intestinal homeostasis requires a continuous dialogue between commensal bacteria and intestinal immune cells. Natural Killer T (NKT) cells are a population of CD1d-restricted lipid-reactive lymphocytes contributing to the regulation of mucosal immunity, but the mechanisms underlying this are poorly understood. Here we show that lipid presentation by CD1d+ intestinal dendritic cells and macrophages controls NKT cell function and activation which in turn regulates commensal bacteria and immune cell populations in the gut. These results reveal an NKT cell-dendritic cell crosstalk as a key mechanism for the regulation of intestinal homeostasis.
Mouse retinal cell behaviour in space and time using light sheet fluorescence microscopy
We successfully performed the first lightsheet 3D/4D imaging of mouse retinas (focussing on vessels and neurons) to demonstrate that current confocal methods distort vessel tissue. This brings a much improved way to observe and quantify the devastating changes to vessels and neurons in retinopathy mouse models.
A protease cascade regulates release of the human malaria parasite Plasmodium falciparum from host red blood cells
This study showed that egress involves an enzyme cascade in which the serine protease SUB1 activates a second, cysteine protease called SERA6, enabling SERA6 to rapidly and precisely cleave the major red cell cytoskeletal protein β-spectrin and dismantle the cytoskeleton. It provides the first plausible model to explain how the parasite accomplishes timely rupture of its host cell membrane.
Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds
In this paper we define the heterogeneity and the clonogenic potential of human thymus stroma; characterise progenitor cells capable of extensive expansion in vitro, thereby achieving clinically relevant numbers with resilience to long-term storage; and report an epithelial-mesenchymal hybrid phenotype of thymus epithelial cells in vivo and in vitro that affects cell behaviour, a unique feature among any epithelia so far reported. We describe a protocol for organs that lack a main vascular access that allowed us to specify the role of natural ECM in supporting organ morphogenesis ex vivo and in vivo; and reconstitute a functional human thymus long-term in vivo.
Stabilization of reversed replication forks by telomerase drives telomere catastrophe
This study defined the mechanism leading to critically short telomeres in the absence of RTEL1 and showed that telomerase, which extends telomeres in normal cells, is pathological when forks encounter an obstacle within the telomere. We showed that replication forks stall and reverse at persistent t-loops, which creates a pseudo-telomere substrate that is inappropriately stabilised by telomerase. Removing telomerase or blocking replication fork reversal rescued telomere dysfunction in Rtel1 deficient cells. We proposed that when persistent t-loops stall the replisome, telomerase inhibits fork restart, triggering the excision of the t-loop by SLX1/4 and loss of a substantial part of the telomere.
Species-specific pace of development is associated with differences in protein stability
Despite evolutionarily conservation of molecular mechanisms, the speed of development varies substantially between species. Using in vitro directed differentiation of embryonic stem cells to motor neurons, we show that the programme of motor neuron differentiation runs twice as fast in mouse as in human. We provide evidence that a two-fold increase in protein stability and cell cycle duration in human cells compared to mouse can account for the slower pace of human development, indicating that global differences in kinetic parameters play a major role in interspecies differences in developmental tempo. This study establishes a new experimental system in which to address fundamental questions.
Nervous system regionalization entails axial allocation before neural differentiation
The prevailing view of neural induction in vertebrate embryos had been that cells are initially induced with anterior (forebrain) identity and then caudalising signals convert a proportion to posterior fates (spinal cord). Using chromatin accessibility, to define how cells adopt region-specific neural fates, combined with genetic and biochemical perturbations, we found that contrary to the established model, cells commit to a regional identity before acquiring neural identity. These findings prompt a revision to textbook models of neural induction. The study illustrates our adoption of new genomic methods (ATACseq) to address long-standing questions, and our capacity to productively collaborate with computational biologists.
Restriction of memory B cell differentiation at the germinal center B cell positive selection stage
Memory B cells (MBCs) are key for protection from reinfection. However, it is mechanistically unclear how germinal center (GC) B cells differentiate into MBCs. MYC is transiently induced in cells fated for GC expansion and plasma cell (PC) formation, so-called positively selected GC B cells. We found that these cells coexpressed MYC and MIZ1 (MYC-interacting zinc-finger protein 1 [ZBTB17]). MYC and MIZ1 are transcriptional activators; however, they form a transcriptional repressor complex that represses MIZ1 target genes. Mice lacking MYC-MIZ1 complexes displayed impaired cell cycle entry of positively selected GC B cells and reduced GC B cell expansion and PC formation. Notably, absence of MYC-MIZ1 complexes in positively selected GC B cells led to a gene expression profile alike that of MBCs and increased MBC differentiation. Thus, at the GC positive selection stage, MYC-MIZ1 complexes are required for effective GC expansion and PC formation and to restrict MBC differentiation. We propose that MYC and MIZ1 form a module that regulates GC B cell fate.
D-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition
D-cycloserine is an antibiotic used for decades to treat drug resistant tuberculosis. Its inhibition mechanism came into question when in a previous paper we determined alanine racemase activity in “fully inhibited” cells. This study demonstrated a previously unknown path during the assumed irreversible inhibition of alanine racemase that leads to the destruction of the antibiotic, meaning that alanine racemase is not irreversibly inhibited by the drug. The paper highlights the complexity of studying the chemical mechanisms of inhibition of enzymes and points to a novel strategy to design D-cycloserine analogues with improved properties.
Structural basis of second-generation HIV integrase inhibitor action and viral resistance
HIV integrase inhibitors represent some of the most impactful antimicrobial inhibitors. The second-generation drugs display improved barriers to the emergence of resistance, which spearheaded their worldwide rollout. Yet not even the most advanced compounds are immune to viral resistance. Our results explained the mechanism of viral resistance associated with the most common drug resistance mutations. Furthermore, we established the key difference between the first and second-generation strand transfer inhibitors, which will inform further development of this drug class.
Pandemic peak SARS-CoV-2 infection and seroconversion rates in London frontline health-care workers
This important paper showed very high levels of infection amongst healthcare workers in a local hospital. It has influenced government policy – asymptomatic healthcare workers are to be screened as per our recommendation (announced October 12th).
The mechanism of eukaryotic CMG helicase activation
This paper provided the first view of how the inactive MCM double hexamer is converted to two active CMG helicases. We showed MCM remains bound to ADP after loading; firing factors trigger ADP-ATP exchange; ATP rebinding causes double hexamer splitting, initial DNA melting and CMG formation. Active helicases then translocate N-terminus first.
Modular microfluidics enables kinetic insight from time-resolved cryo-EM
Cryo-EM has the potential to study any native conformation of a macromolecule. However, the sample preparation time is high, compared to the timescale of most protein interactions and conformational changes. In this paper, we established a robust method of time-resolved cryo-EM sample preparation. We produced high-quality samples for microscopy while speeding up the process of making them by several orders of magnitude. This allowed samples to be collected within 30ms of the initiation of a biochemical reaction, within the timeframe of many critically important and interesting processes. This enables a whole new class of experiments in structural biology research.
SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects
We have been able to apply the knowledge we have gained from our work on the infectivity of the influenza virus to the challenge presented by the recent SARS-CoV-2 virus outbreak. In this paper we present high resolution cryo EM structures of the SARS-CoV-2 and bat RaTG13 spike glycoproteins. We describe from a structural perspective the significant differences between the strains. We draw particular attention to the addition of a furin cleavage site into the human virus spike protein. We discuss its potential role in infectivity and on the evolution of this virulent strain.
Published in Nature Structural & Molecular Biology
Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion
Here we describe the conformational changes that the SARS-Cov2 spike protein undergoes in binding to the human ACE2 receptor. This represents the initial stages of the mechanism of cell invasion by the virus particle during infection. We show a series of ten cryoEM reconstructions of the spike protein binding to ACE2 through its receptor binding domain (RBD), ranging from a closed unbound spike ectodomain trimer to the fully open conformation with each RBD in the trimer bound to an ACE2 receptor. Binding to ACE2 releases the so-called fusion peptide segment and promotes membrane fusion leading to cell invasion.
α-synuclein oligomers interact with ATP synthase and open the permeability transition pore in Parkinson’s disease
Protein aggregation drives neuronal death in Parkinson’s disease, although how transition of monomeric protein structures to aggregated forms causes toxicity is unknown. We demonstrate that aggregation of the protein α-synuclein generates beta sheet-rich oligomers, which localise to the mitochondrial inner membrane, where they impair complex I-dependent respiration, induce oxidation of ATP synthase and cause mitochondrial lipid peroxidation. These oxidation events result in opening of the permeability transition pore, triggering mitochondrial swelling, and ultimately cell death. This work highlights how structural conversion of a protein changes its physiological interaction with proteins and lipids, and induces pathology in human cell models of disease.
Alpha synuclein aggregation drives ferroptosis: an interplay of iron, calcium and lipid peroxidation
Aberrant protein-lipid interactions occur in neurodegeneration, although their role is unclear. We show how the protein α-synuclein interacts with lipids to drive a form of cell death, ferroptosis. As α-synuclein aggregates, oligomeric species with hydrophobic domains incorporate into the plasmalemmal membrane, leading to altered membrane conductance and abnormal calcium influx following glutamatergic and dopaminergic stimuli. Aggregates induce iron dependent generation of free radicals, and peroxidation of polyunsaturated fatty acids, which underlies the incorporation of aggregates into the membranes. Targeted inhibition of lipid peroxidation prevents the aggregate-membrane interaction, abolishes aberrant calcium fluxes, and restores physiological calcium signaling in human neurons, highlighting a new causative role for lipid homeostasis in Parkinson’s disease.
Early-life exposure to low-dose oxidants can increase longevity via microbiome remodelling in Drosophila
This reports the first identification, in any species, of the microbiome as a key mediator of developmental stress-induced longevity. We found that mild oxidative stress during development robustly increases lifespan via the selective elimination of Acetobacter from the microbiome. This study also highlights that targeted remodelling of the early-life microbiome can provide an efficient strategy for extending healthspan and lifespan.
High-throughput phenotyping reveals expansive genetic and structural underpinnings of immune variation
The immune system is increasingly acknowledged to be integrated with general physiology, but the genetic pathways underpinning those are largely unknown. This study demonstrated that high-content immunophenotyping could be accomplished at scale, compatible with a genetic screen and in so doing identified 80 novel immunoregulators (“hits”) and established striking correlations of immunological traits with blood biochemistry markers such as cholesterol and sodium. The paper formed a basis for the successful and rapid application of high-content high-throughput profiling to COVID-IP and to cancer immunomonitoring, and has spawned mechanistic follow-up studies of several of the hits.