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.
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.
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.
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.
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).
Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation
Here we showed that Sld3, which we previously identified as being one of two essential cyclin dependent kinase (CDK) substrates in replication, is a phosphopeptide binding protein which binds specifically to Mcm4 and Mcm6 when they have been phosphorylated by Dbf4 dependent kinase (DDK). Sld3 then directly recruits Cdc45 to MCM and, via CDK phosphorylation, recruits the remaining firing factors. We had previously shown that Sld3 is also one of two targets of the DNA damage checkpoint kinase involved in inhibiting origin firing in response to DNA damage. Thus, Sld3 plays key roles with all three kinases that regulate replication (CDK, DDK, Rad53).
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.
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.
Return to quiescence of mouse neural stem cells by degradation of a proactivation protein
This paper provided the first evidence that stem cells in the adult mouse hippocampus are heterogeneous in their behaviour, with most stem cells differentiating and leaving the niche after they have become active but a small fraction returning to a shallow state of quiescence. These “resting cells” have an essential role in the long-term maintenance of an active stem cell pool.
Epithelia use butyrophilin-like molecules to shape organ-specific γδ T cell compartments
This paper established that intestinal epithelial cells use BTNL/Btnl molecules to select for and regulate tissue-specific gamma delta T cell compartments. It established a biological mechanism by which epithelial cells communicate with local T cells at steady-state (“normality sensing”). Following on from our prototypic discovery of such a mechanism in mouse skin, the work established conservation of the process across tissues as well as across species. The system is unperturbed by microbial colonisation.
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.
A dynamic COVID-19 immune signature includes associations with poor prognosis
SARS-CoV-2 infection and life-threatening COVID-19 caused the world’s most severe infectious disease pandemic in 100 years. An immediate priority was to decipher what was happening to patients’ immune systems. Rapidly deploying its skill-sets in high-content, high-throughput immunoprofiling, the Immunosurveillance Laboratory identified a dynamic, COVID-19 immune signature that blended textbook immunoprotection with examples of immune dysregulation that today’s textbooks do not describe. Among those, three molecules measured upon hospital admission seemingly predict a patient’s likelihood of deterioration over the next week; knowledge which can benefit health-care resource management, and offer novel therapeutic targets in COVID-19 and other inflammatory infectious diseases.
Ubiquitin activation is essential for schizont maturation in Plasmodium falciparum blood-stage development
This study describes the ubiquitome of several stages of the intra-erythrocytic development and extracellular stage of the malaria parasite in the blood stream. It highlights the remarkable changes in ubiquitylation that occur and a number of very interesting substrates. Using a chemical biology approach we show the importance of the first step in the pathway and the consequences of its inhibition during intra-erythrocytic development.
Preexisting and de novo humoral immunity to SARS-CoV-2 in humans
An example of our work on COVID-19 and of the flexible and collaborative nature of the Crick, involving several labs within the Crick and our collaborating universities and university hospitals. In this work, we described the discovery of pre-existing binding and neutralising antibodies against SARS-CoV-2 in uninfected and unexposed individuals. These antibodies, likely induced by exposure to seasonal coronaviruses, are present in a small percent of adults but in the majority of children, consistent with the relative sparing of the latter from the severe form of COVID-19
Engineering transplantable jejunal mucosal grafts using patient-derived organoids from children with intestinal failure
Children with intestinal failure cannot absorb the nutrients that are essential to be healthy. In the most severe cases, patients may require transplantation. However, there is a shortage of donor organs and complications can arise after surgery. We have shown how intestinal stem cells and intestinal tissues taken from patients can be used to grow functioning intestinal grafts in the laboratory, which could offer a safe and longer-lasting alternative to traditional donor transplants.