Speakers

The ABC of TDP-43 

Abnormal assemblies of TDP-43 in neurons and glia are the pathological hallmark of amyotrophic lateral sclerosis (ALS) and multiple types of frontotemporal lobar degeneration (FTLD).  Mutations in the TDP-43 gene, TARDBP, can cause ALS and FTLD, and the temporospatial accumulation of TDP-43 assemblies correlates with neurodegeneration, indicating a causative role for TDP-43 assembly in disease. TDP-43 assemblies are also common co-pathologies in other diseases, including Alzheimer’s, Parkinson’s and Huntington’s. The structural and molecular mechanisms of TDP-43 assembly in disease are poorly understood. We developed a protocol to isolate assembled TDP-43 from the brains of patients with ALS and FTLD and determined their structures using cryo-electron microscopy (cryo-EM). We found that TDP-43 assembles into amyloid filaments in these diseases. The ordered filament cores are comprised of the first half of the TDP-43 low-complexity domain and adopt distinct filament folds in different neurodegenerative conditions. These brain-derived filament folds show no similarity to TDP-43 filament folds formed in vitro. The structures, in combination with mass spectrometry, led to the identification of two new post-translational modifications of assembled TDP-43, citrullination and mono-methylation of R293, and suggest that they may facilitate filament formation and observed structural variation within individual filaments. Unexpectedly, the structures also revealed that in specific cases TDP-43 can also co-assemble with Annexin A11 in heteromeric amyloid filaments. The structures of TDP-43 amyloid filaments from ALS and FTLD guide mechanistic studies of TDP-43 assembly, as well as the development of diagnostic and therapeutic compounds for TDP-43 proteinopathies.

How microbes control our immune system? 

Our immune system is regulated at barrier surfaces to protect against pathogens – and promote tissue homeostasis – in the presence of commensal microbes, food antigens and other environmental stimuli. Considering the host as a metaorganism has profoundly impacted our understanding of host physiology and immunity, from the response to vaccinations to  protection against infections. 

Studying the structure and maturation of HIV-1 using cryo-electron microscopy

Cryo-electron microscopy, cryo-electron tomography and computational image processing can be combined to determine the structures of proteins within complex environments like cells and irregular viruses. We are applying these techniques to understand how proteins interact with one another and with lipids to assemble enveloped viruses and coated vesicles, and to understand how the proteins then rearrange to adapt their functions at different stages of the virus or vesicle lifecycle. Retroviruses like HIV-1 are initially produced as immature, non-infectious particles with the main structural protein Gag forming a membrane-associated protein layer. Cleavage of Gag separates individual domains leading to morphological conversion into the mature, infectious virion. I will describe how we are using cryo-electron microscopy techniques to study virus morphology, to obtain structures of HIV-1 proteins from within immature, mature and mutant HIV-1 particles, and to reconstitute aspects of virus assembly in vitro. Together, these data lead us closer to a mechanistic understanding of the late stages of the HIV-1 lifecycle. 

How DNA replication initiates and what happens when it goes wrong 

The eukaryotic cell cycle coordinates the accurate duplication and segregation of the genome during proliferation.  Central to this is the mechanism that ensures the entire genome is precisely duplicated in each cell cycle.  The large genomes of eukaryotic cells are replicated from multiple replication origins during S phase.  These origins are not activated synchronously at the beginning of S phase, but instead fire throughout S phase according to a pre-determined, cell type specific program.  Yet each of these origins must be regulated to ensure they never fire more than once per cell cycle.  In addition to duplication of genomic DNA, all of the protein components of chromosomes must be duplicated during S phase so gene expression patterns can be maintained.  Replication must also be coordinated with many nuclear processes including sister chromatid cohesion, replication checkpoint activation, postreplication repair and chromatin assembly. Once per cell cycle regulation of replication origin firing is achieved by a two-step mechanism.  The first step involves the loading of the Mcm2-7 proteins into pre-replicative complexes (pre-RCs) at origins. In the second step, the MCM helicase is activated by a large set of ‘firing factors’. These two steps are differentially regulated by cyclin dependent kinase (CDK): MCM loading is inhibited by CDK, whilst firing requires CDK. As a consequence, MCM loading can only happen during G1 phase, when CDK activity is low, and origin firing cannot occur during G1 phase.  Other kinases, like the Dbf4 dependent kinase (DDK) and the Rad53 checkpoint kinase also directly regulate replication.  Misregulation of this system can generate chromosome rearrangements which may contribute to genome instability in cancer. We use a combination of approaches including biochemistry, structural biology, genetics and cell biology to understand how DNA replication initiates, how replication forks are established, how epigenetic information encoded in parental nucleosomes is inherited and how the DNA damage checkpoint contributes to replication fork stability. 

Hair follicles. What’s sensory got to do with it? 

Human hair follicle epithelial stem cells (HFESCs) are supported by an elegant perineural niche comprised of highly organised sensory and sympathetic nerve endings. Although recent studies have demonstrated the importance of these nerves in regulating several aspects of skin and hair follicle biology, including hair cycling, pigmentation, and wound healing, how HFESCs might influence sensory nerve biology and function in a two-way manner is poorly understood. We recently showed that in vitro, human HFESCs are mechanosensitive and relay sensory information to mouse dorsal root ganglia sensory neurons, activating them via the vesicular release of neurotransmitters (serotonin and histamine) in a co-culture system. In this presentation, I will discuss the additional functional roles that HFESC and HFESC derived neurotransmitters may carry out in the contexts of skin and nervous system development, homeostasis, and regeneration

Phagocytes: Hungry After an All-You-Can-Eat Buffet – Maintaining Function Amidst Continuous Clearance  

Phagocytic cells, such as macrophages, microglia, and retinal epithelial cells, are responsible for internalising and degrading vast quantities of diverse extracellular material, including dead cells, lipoproteins, synapses, microorganisms, and photoreceptor outer segments. Remarkably, the human body clears over 200 billion dead cells each day. In doing so, phagocytes must maintain their health and functionality, as failure to properly clear and process internalised cargo can lead to a range of diseases, including atherosclerosis, infection, autoimmunity, macular degeneration, and neurodegeneration. Understanding how phagocytes efficiently manage such large, diverse loads within intracellular compartments is crucial for addressing fundamental questions in cell biology and understanding the molecular mechanisms underlying disease. Some of the key questions we aim to answer: (i) What is the fate of degraded cargo, and how is it mobilised within the cell?  (ii) How do phagocytes restore their resting state after phagocytosis? (iii) What are the consequences of impaired clearance of extracellular materials? 

Unveiling the Complexities of Cellular Mechanisms: Beyond Protein Expression 

Living systems operate through complex regulatory networks that extend beyond simple protein expression. These networks incorporate multiple sophisticated multi-omics control layers, from non-coding RNA-mediated control mechanisms, post-transcriptional and post-translational modifications, to differential subcellular localization. Many of these regulatory processes depend on dynamic interactions between proteins and RNA molecules. Understanding these multilayered control systems is essential for gaining complete insight into how biological processes function. This presentation will highlight why examining how the proteome responds to cellular perturbations through protein relocalization is critical, demonstrating that focusing solely on abundance changes can cause researchers to miss essential cellular responses¹. I will examine how RNA-protein interactions shift dynamically during pivotal cellular events²,³. I will also introduce an innovative methodology that simultaneously maps both the spatial distribution of proteins and RNA transcripts across entire cells⁴. I will show how this approach has uncovered the coordinated movement of RNA and proteins when cells activate the unfolded protein response (UPR), offering new understanding of how RNA molecules relocate to stress granules during cellular stress. 

References: 

1. Christopher, J. A. et al doi: 10.1016/j.mcpro.2024.100888. 

2. Queiroz, R. M. L et al doi:10.1038/s41587-018-0001-2  

3. Monti, M et al doi:10.1038/s44320-024-00031-y  

4. Villanueva, E et al S. doi:10.1038/s41592-023-

Targeted cellular evolution identifies new sensory receptor proteins 

Gene identification by conventional screening means poses many challenges. This includes cost and labour requirements of arrayed screening and limitations imposed by library quality and reliance on genome annotation data. Forward screening approaches, such as cDNA- or CRISPRa-induced gene expression, are limited in scope to single-gene phenotypes. Reverse screening techniques, such as RNAi or CRISPRi-mediated knockdown, can identify genes functioning in multi-protein complexes, but are limited by the availability of a cell line already expressing the positive phenotype. We combined a random forward mutagenesis approach with unique fluorescent selection tools to develop a screening approach capable of reliable, high-yield and cost-effective gene identification. Our approach combines the strengths of forward and reverse screening: it enables identification of multi-gene receptors in an array of phenotypically negative cell environments, and bypasses library quality-related bias by utilising the endogenous genome. The efficiency of this method is demonstrated through confirmation of known, and identification of novel molecular temperature receptors in a single experiment. Targeted cellular evolution thus provides the capability of unbiased, massively parallel screening to identify novel receptor genes where no cellular survival-based selection pressure is available. 

Investigation of the HR1 domains of protein kinase C-related kinase (PKN1) 
 
The PKN family are Ser/Thr kinases that are activated downstream of Rho family small GTPases. PKN protein activation has been implicated in tumours, particularly prostate (PKN1) and breast (PKN3) cancer. PKN1 is involved in insulin signalling to the actin cytoskeleton, in phosphorylation of histone H3 in androgen receptor signalling and in regulation of the pyrin inflammasome. All PKN proteins have the same domain structure, with three HR1 domains in the N-terminus, followed by a C2 domain and a C-terminal kinase domain. PKN is activated by Rho protein binding, by PDK1 phosphorylation or by caspase cleavage of the N-terminus and is inhibited by binding to a short sequence between the C2 and kinase domain. Structural data on the protein is limited to single HR1 domains and to the kinase domain alone. We have used a combination of NMR, SAXS and biophysical methods to study the structures and interactions of larger constructs of PKN1 that include multiple HR1 domains. We have found that the HR1 domains are a dimerization motif, and that this dimer is rearranged upon RhoA binding. PKN1 dimers were observed in cell experiments and could be disrupted by mutations in HR1 domains. Work is undergoing to assess the effect of the mutants on kinase activity. 

Yersinia pestis & plague: revisiting the history and genomic landscape of a pandemic tueur 

In 1894 in Hong Kong, Louis Pasteur’s collaborator Alexandre Yersin identified Yersinia pestis as the bacterial agent responsible for plague, a mythical disease which had killed millions of human beings in the course of several pandemics, leaving deep scars on the history of humanity. Since then, the Pasteurian school of microbiology has made significant contributions to our understanding of plague, identifying its transmission mechanisms, contributing to the development of vaccines and improving diagnostics. Today, the revolution in ancient DNA research enables us to revisit plague pandemics of historic and also prehistoric times, to study the impact of medieval plague on the evolution of the human immune system, and to investigate molecular and cellular mechanisms of plague pathogenesis. In the current post-COVID context, plague remains a fascinating disease that still has much to teach us about the emergence and evolution of pathogens. 

Euk.oli: A eukaryotic-inspired prokaryotic model for orthogonal resource allocation 

Resource competition between native and synthetic genes imposes a burden on the cell which leads to growth defects and unpredictable synthetic gene circuit and pathway behaviour. Importantly, this resource competition problem, especially at the level of translation, seriously impedes the development of synthetic biology as a field. To better understand this issue, our previous work focused on quantitatively understanding trade-offs between bacterial growth and the allocation of cellular resources for gene expression. Through this research, we identified rRNA expression as a critical regulatory node and devised engineering strategies to uncouple rRNA synthesis from the overall physiological status of the model bacterium Escherichia coli. Based on this research and inspired by the distinct transcriptional control of rRNA in eukaryotes, we have engineered a set of E. coli strains —named Euk.oli— in which all chromosomal rRNA operons were removed, and rRNA expression takes place from a plasmid as is placed under the control of inducible promoters and, in a further engineered version, an orthogonal T7 RNA polymerase. This approach mimics to an extent the eukaryotic strategy of allocating different RNA polymerases for mRNA and rRNA synthesis. As a result, Euk.oli strains exhibit unique properties: their growth rates are directly correlated with rRNA synthesis, and they demonstrate enhanced production of recombinant proteins and metabolic pathways for high-added value molecules.