Seminars

In recent years, it has become clear that intrinsically disordered regions (IDRs) of RBPs, and the structure of RNAs, often contribute to the condensation of RNPs. To understand the transcriptomic features of such RNP condensates, we’ve used an improved individual nucleotide resolution CLIP protocol (iiCLIP), which produces highly sensitive and specific data, and thus enables quantitative comparisons of interactions across conditions (Lee et al., 2021). This showed how the IDR-dependent condensation properties of TDP-43 specify its RNA binding and regulatory repertoire (Hallegger et al., 2021). Moreover, we developed software for discovery and visualisation of RNA binding motifs that uncovered common binding patterns of RBPs on long multivalent RNA regions that are composed of dispersed motif clusters (Kuret et al, 2021). Finally, we used hybrid iCLIP (hiCLIP) to characterise the RNA structures mediating the assembly of Staufen RNPs across mammalian brain development, which demonstrated the roles of long-range RNA duplexes in the compaction of long 3’UTRs. I will present how the combined analysis of the characteristics of IDRs in RBPs, multivalent RNA regions and RNA structures is required to understand the formation and functions of RNP condensates, and how they change in diseases.

CA3-CA1 synapses in the hippocampus are the initial locus of episodic memory. The action of acetylcholine alters cellular excitability, modifies neuronal networks, and triggers secondary signaling that directly affects long-term plasticity (LTP) (the cellular underpinning of memory). It is therefore considered a critical regulator of learning and memory in the brain. Its action via M4 metabotropic receptors in the presynaptic terminal of the CA3 neurons and M1 metabotropic receptors in the postsynaptic spines of CA1 neurons produce rich dynamics across multiple timescales. We developed a model to describe the activation of postsynaptic M1 receptors that leads to IP3 production from membrane PIP2 molecules. The binding of IP3 to IP3 receptors in the endoplasmic reticulum (ER) ultimately causes calcium release. This calcium release from the ER activates potassium channels like the calcium-activated SK channels and alters different aspects of synaptic signaling. In an independent signaling cascade, M1 receptors also directly suppress SK channels and the voltage-activated KCNQ2/3 channels, enhancing post-synaptic excitability. In the CA3 presynaptic terminal, we model the reduction of the voltage sensitivity of voltage-gated calcium channels (VGCCs) and the resulting suppression of neurotransmitter release by the action of the M4 receptors. Our results show that the reduced initial release probability because of acetylcholine alters short-term plasticity (STP) dynamics. We characterize the dichotomy of suppressing neurotransmitter release from CA3 neurons and the enhanced excitability of the postsynaptic CA1 spine. Mechanisms underlying STP operate over a few seconds, while those responsible for LTP last for hours, and both forms of plasticity have been linked with very distinct functions in the brain. We show that the concurrent suppression of neurotransmitter release and increased sensitivity conserves neurotransmitter vesicles and enhances the reliability in plasticity. Our work establishes a relationship between STP and LTP coordinated by neuromodulation with acetylcholine.

Diseases that arise from misfolding of an individual protein are rare. However, collectively, these folding diseases represent a large proportion of hereditary and acquired disorders. In fact, the term "Molecular Medicine" was coined by Linus Pauling in conjunction with the study of a folding disease, i.e. sickle cell anemia. In the past decade, we have witnessed an exponential growth in the number of mutations, which have been identified in genes encoding solute carriers (SLC). A sizable faction - presumably the majority - of these mutations result in misfolding of the encoded protein. While studying the export of the GABA transporter (SLC6A1) and of the serotonin transporter (SLC6A4), from the endoplasmic reticulum (ER), we discovered by serendipity that some ligands can correct the folding defect imparted by point mutations. These bind to the inward facing state. The most effective compound is noribogaine, the metabolite of ibogaine (an alkaloid first isolated from the shrub Tabernanthe iboga). There are 13 mutations in the human dopamine transporter (DAT, SLC6A3), which give rise to a syndrome of infantile Parkinsonism and dystonia. We capitalized on our insights to explore, if the disease-relevant mutant proteins were amenable to pharmacological correction. Drosopohila melanogaster, which lack the dopamine transporter, are hyperactive and sleepless (fumin in Japanese). Thus, mutated human DAT variants can be introduced into fumin flies. This allows for examining the effect of pharmacochaperones on delivery of DAT to the axonal territory and on restoring sleep. We explored the chemical space populated by variations of the ibogaine structure to identify an analogue (referred to as compound 9b), which was highly effective: compound 9b also restored folding in DAT variants, which were not amenable to rescue by noribogaine. Deficiencies in the human creatine transporter-1 (CrT1, SLC6A8) give rise to a syndrome of intellectual disability and seizures and accounts for 5% of genetically based intellectual disabilities in boys. Point mutations occur, in part, at positions, which are homologous to those of folding-deficient DAT variants. CrT1 lacks the rich pharmacology of monoamine transporters. Nevertheless, our insights are also applicable to rescuing some disease-related variants of CrT1. Finally, the question arises how one can address the folding problem. We propose a two-pronged approach: (i) analyzing the effect of mutations on the transport cycle by electrophysiological recordings; this allows for extracting information on the rates of conformational transitions. The underlying assumption posits that - even when remedied by pharmacochaperoning - folding-deficient mutants must differ in the conformational transitions associated with the transport cycle. (ii) analyzing the effect of mutations on the two components of protein stability, i.e. thermodynamic and kinetic stability. This is expected to provide a glimpse of the energy landscape, which governs the folding trajectory.

Biomolecular condensation is a mechanism for controlling cell organization. Many condensates are rich in nuclei acids such as RNA. The role of specific RNA sequences and structures in promoting the molecular identity of condensates formed for cell polarity and division and by the SARS CoV-2 virus will be discussed.

My lab studies the design principles of cytoskeletal materials the drive cellular morphogenesis, with a focus on contractile machinery in adherent cells. In addition to force generation, a key feature of these materials are distributed force sensors which allow for rapid assembly, adaptation, repair and disintegration. Here I will discuss our recent identification of 18 proteins from the zyxin, paxillin, Tes and Enigma families with mechanosensitive LIM (Lin11, Isl- 1 & Mec-3) domains. We developed a screen to assess the force-dependent localization of LIM domain-containing region (LCR) from ~30 genes to the actin cytoskeleton and identified features common to their force-sensitive localization. Through in vitro reconstitution, we found that the LCR binds directly to mechanically stressed actin filaments. Moreover, the LCR from the fission yeast protein paxillin-like 1 is also mechanosensitive, suggesting force-sensitivity is highly conserved. We speculate that the evolutionary emergence of contractile F-actin machinery coincided with, or required, proteins that could report on the stresses present there to maintain homeostasis of actively stressed networks.

Evidence regarding protein structure and function manifest the imperative role that dynamics play in proteins, underlining reconsideration of the unanimated sequence-to-structure-to-function paradigm. Structural dynamics portray a heterogeneous energy landscape described by conformational ensembles where each structural representation can be responsible for unique functions or enable macromolecular assemblies. Using the human p27/Cdk2/Cyclin A ternary complex as an example, we highlight the vital role of intra- and intermolecular dynamics for target recognition, binding, and inhibition as a critical modulator of cell division. Rapidly sampling configurations is critical for the population of different conformational ensembles encoding functional roles. To garner this knowledge, we present how the integration of (sub)ensemble and single-molecule fluorescence spectroscopy with molecular dynamic simulations can characterize structural dynamics linking the heterogeneous ensembles to function. The incorporation of dynamics into the sequence-to-structure-to-function paradigm promises to assist in tackling various challenges, including understanding the formation and regulation of mesoscale assemblies inside cells.

This talk aims to show how new physical methods can advance biological and biomedical research. A major advance in physical chemistry in the last two decades has been the development of quantitative methods to directly observe individual molecules in solution, attached to surfaces, in the membrane of live cells or more recently inside live cells. These single-molecule fluorescence studies have now reached a stage where they can provide new insights into important biological problems. After presenting the principles of these methods, I will give some examples from our current research to probe the molecular basis of neurodegeneration. Here we have used single-molecule fluorescence to detect and analyse the low concentrations of soluble protein aggregates thought to be responsible for Alzheimer’s disease and determine the mechanisms by which they damage neurons. Lastly, I will describe how fundamental science aimed at watching single molecules incorporating nucleotides into DNA gave rise to a new rapid method to sequence DNA that is now widely used.