Organisms form crystalline materials with superior structural and mechanical properties. This arises from the ability of functional macromolecules to create intricate architectures via a multi-step crystallization process. Current approaches to engineer bioinspired minerals focus on interactions between macromolecules and minerals in dilute aqueous environments, rarely considering the emergent properties of macromolecular condensates. However, we and others showed that macromolecular crowding is intimately associated with biomineral formation in vivo. In this project, we will develop a new type of chemistry—dense-phase mineralization—to unlock the pathways mastered by nature. Our hypothesis is that weak polymer-ion interactions within dense phases tune the chemical landscape, controlling the crystallization process and the properties of its products. Remarkably, our preliminary results using the calcium carbonate system show that molar-range polymer concentrations, four orders of magnitude denser than in previous works, result in intricate crystals with life-like properties. We will investigate dense-phase mineralization in both synthetic and living systems, relying on our unique expertise in cryo-electron and X-ray microscopies of hydrated biological samples. In Aim 1, we will grow crystals in a dense polymer phase and use the crowded environment to sculpt architectural motives. In Aim 2, we will investigate the challenging phase separation regime and transform inorganic condensates into transient precursors for mineralization. In Aim 3, we will elucidate how liquid-liquid phase separation evolved by mineralizing organisms to regulate inorganic condensate formation. This project will open an uncharted chemical landscape to form and control bioinspired minerals. The outcome will be a toolbox for process design that allows to optimize material properties - the highest gain we can ask for in bioinspired mineralization.

A remarkable diversity of post-transcriptional modifications adorn the ribosomal RNA (rRNA) throughout all domains of life. While initially conceptualized as a constitutive component of the ribosome, recent evidence demonstrates that some modifications are only present on a fraction of cellular ribosomes, and our lab recently revealed unprecedented environmentally-mediated dynamics of an rRNA modification. This suggests that some rRNA modification may serve as a tunable layer for regulation of ribosome properties, facilitating adaptation to a new environment, stress or pathological state. Here we aim to systematically explore the extent to which rRNA modifications are tunable across evolution and highly diverse growth conditions, and to understand the consequences thereof. We seek to understand which rRNA modifications are subject to regulation, to uncover the contexts in which such regulation occurs, and to unravel the underlying mechanisms. Dissecting these questions requires an approach allowing systematic measurement of diverse rRNA modifications. We propose to establish a streamlined workflow, allowing multiplexed, precise, robust and cost-efficient systematic profiling of 19 distinct rRNA modifications in dozens of samples in a single experiment (Aim 1). In Aim 2, we will systematically measure rRNA modifications across the three domains of life, focusing on species that can thrive across two widely different physical/chemical gradients, where the potential for tunable rRNA modifications is particularly high. In Aim 3, we will combine gain- and loss-of-function approaches to systematically explore the functions RNA modifications can bestow on ribosomes. Collectively, EpiRibo addresses a fundamental open question regarding the extent of plasticity and regulatory potential present within the ribosome itself and encoded via its epitranscriptome, setting the stage for investigations in other RNA species, and in more complex and disease-related contexts.

Cryo-electron microscopy has revolutionized the field of structural biology, primarily for macromolecular structure but also for cells and tissue sections—achieving resolutions at the limit of physical optics. While wide-field transmission EM (TEM) with phase contrast by defocus is the most commonly used modality in biology, the alternative, scanning transmission EM (STEM), has emerged as the mode of choice for atomic resolution in materials science. Seeking to endow biology with the benefits of STEM, our lab established STEM for cryo-tomography of biological cells and demonstrated its advantages for thick specimens and compositional contrast. We now seek to extend cryo-STEM to high-resolution, with an emphasis on tomography, by means of coherent detection (Obj1). This will be achieved by the method of integrated differential phase contrast (iDPC) using a segmented detector, from which we obtain simultaneously phase and depth contrast in a single scan. The major expected benefits are: 1) minimization of image aberration, especially defocus with its associated complications for image interpretation, 2) reduction of beam-induced radiation damage by means of flexible scan and sampling patterns, and 3) improved reconstruction for tomography based on tailored data acquisition. We will validate the new methods for single particle analysis on standard macromolecular substrates and compare them to current state-of-the-art methods. Further, we will apply the new developments in 3D imaging to explore novel large-scale structures in chromatin we observed recently by whole-cell cryo-STEM tomography using current, low-resolution methods (Obj2). Labelling with halogenated nucleotides will reveal sites of active transcription or DNA synthesis. The proposed approaches’ expected broad applicability and STEM’s unrealized potential for hardware simplicity should together ensure the wide adoption of cryo-STEM methods in biology, accelerated by our dissemination efforts (Obj3).

How does experience alter the functional architecture of synaptic connections in neural circuits? This question is particularly pertinent for the complex circuits of the medial prefrontal cortex (mPFC), a high-order associative neocortical area that plays a crucial role in flexible, goal-directed behavior. The mPFC is densely interconnected with cortical and subcortical circuits, and its neurons were shown to undergo substantial experience-dependent structural remodeling that is thought to support learning and memory consolidation. However, little is known regarding the synaptic organization of this complex circuit, and of the functional implications of its experience-dependent structural remodeling. In this proposal, we aim to uncover the organization and learning-associated dynamics of functional connectivity in the mouse mPFC. To obtain high-resolution maps of cell type-specific synaptic connectivity in the mPFC, we will combine single-cell optogenetic manipulation with calcium imaging and electrophysiology in vitro, and establish the circuit-wide organization of connectivity within and between defined projecting neuron populations. We will test the hypothesis that pyramidal neurons projecting to subcortical targets form tightly interconnected subnetworks, and that inhibitory inputs to these networks, through selective innervation, can modulate information output from the mPFC. To understand how learning changes the functional synaptic organization of the mPFC, we will establish an all-optical system for interrogation of synaptic connectivity in vivo. We will utilize this powerful platform to test the hypothesis that prefrontal-dependent learning is associated with reorganization of local-circuit functional connectivity among identified subcortically-projecting cell assemblies. Our innovative technology will be widely applicable for neural circuit analysis in a variety of systems, and allow us to gain new insights into the complex circuitry of the mPFC.