Imported from Grants Tool 4700675

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Phenylalanine-glycine nucleoporins (FG Nups) are intrinsically disordered proteins that generate the permeability barrier within nuclear pore complexes (NPCs). NPCs are remarkable sorting machines that mediate nucleocytoplasmic transport (NCT) in eukaryotic cells. On one hand, NCT is rapid and selective for cargo-carrying nuclear transport receptors termed karyopherins (Kaps). On the other, the permeability barrier obstructs the passage of non-specific cargoes. Importantly, NPC function is underscored by, amongst others, neurodegenerative disorders and viral pathogenesis that are linked to FG Nup/Kap dysfunction. Despite being central to NPC function, we do not understand the spatiotemporal behavior of the FG Nups in the NPC and the permeability barrier remains highly debated. Due to their conformational flexibility, a structural characterization of the permeability barrier remains lacking and lags significantly behind advances in our understanding of NPC scaffold structure. Likewise, it remains unclear how Kap-cargo interactions with the FG Nups might alter the behavior of the permeability barrier to traverse the NPC. This is further related to the question of whether the permeability barrier plays a role in influencing large-scale conformational changes in the NPC such as to accommodate large cargoes. In this work, we will tackle these two major themes: (i) FG Nup dynamics within the NPC permeability barrier; and (ii) to explore its links to conformational changes in the NPC. To do so, we will employ high-speed atomic force microscopy (HS-AFM) to investigate the permeability barrier within NPCs isolated from S. cerevisiae (budding yeast) nuclei at the single NPC level, at transport-relevant length scales (nm) and timescales (~100 ms). Specifically we will characterize FG Nup dynamic behavior in the absence and presence of Kap-cargo complexes in both native NPCs and ÄFG mutant NPCs. In addition, we will evaluate how the permeability barrier might act as a mechanosensor that induces large-scale conformational changes in the NPC. This will involve a systematic study using different ÄFG mutant NPCs that exhibit different degrees of FG Nup cross-linking within their respective permeability barriers. On this basis, we hypothesize that disrupting inter-FG Nup interactions (e.g., by FG-domain deletions, amphipathic alcohols, large cargo complexes, etc.) facilitates pore dilation by reducing the amount of tension imposed by the FG Nups on the NPC scaffold. Finally, we will substantiate our NPC-level findings at the individual FG Nup-level by investigating the permeability barrier generated by FG Nups tethered within artificial nanochannels (also termed NPC mimics).

Type 6 secretion systems (T6SS) are harpoon-like nanomachines that Gram-negative bacterial cells employ to kill other bacterial and eukaryotic cells.Briefly, the T6SS weaponry is tethered to the bacterial cell envelope by a membrane complex, that serves as a platform upon which a baseplate, an extended spring-like sheath and a central spike are assembled. Sheath contraction is biochemically triggered and results in a rapid ejection of the central spike that pierces through a neighboring cell membrane to deliver toxins and other effectors into it. While fluorescence imaging and structural methods have provided deep insight into T6SS structure and function, its destructive mode of action remains unresolved. Here, we will use high-speed atomic force microscope (HS-AFM) imaging, as well as AFM indentation-type force spectroscopy and confocal microscopy (CM) to study the nanomechanical basis by which the T6SS spike punctures bacterial and eukaryotic cell membranes.

Mechanobiology addresses the crosstalk between the mechanical function of cells and the biochemical reactions that drive them. Some key examples include the separation of sister chromatids by the spindle apparatus during cell division, reorganization of the cytoskeleton in somatic cells under mechanical stress, and transformation of fibroblasts into stem-cell like states due to physical confinement. Yet, in spite of being fundamental to cells, intracellular forces remain poorly resolved. This is due in part to a lack of non-invasive methods that facilitate such measurements. In this project, we will develop nanoscale acsoutic tweezers (ATZs) for non-invasive intracellular manipulation. ATZs will be applied to study the impact of mechanical deformation of intra-cellular organelles in-situ; for example, the cell nucleus.

Cells sense and respond to their surroundings using a complex interplay of mechanical forces and biochemical interactions. Mechanical forces that impinge on the cell are transduced from membrane receptors to the extracellular matrix and into the nucleus as one mechanically coupled system. This process activates essential mechanoresponsive transcription factors (MTFs) that are imported into the cell nucleus to modulate gene expression. Although the force-transducing mechanisms are generally understood, little is known as to how mechanical loading and deformation can impact on the ability for a cell to selectively deliver MTFs into the nucleus. This proceeds through highly selective channels in the nuclear envelope known as nuclear pore complexes (NPCs). In part, this work is technically challenging because of the crosstalk between mechanical and biochemical attributes that act simultaneously in space and time. Here, we will develop a correlative multimodal imaging method (CMI) known as the Mechano-Optical Microscope (MOM) that synchronizes atomic force microscope and spinning disk confocal microscope data acquisition within a single integrated platform. In doing so, the MOM will provide both mechanical and biochemical views of biological functionality in live cells. This includes quantitative, multidimensional information with respect to force-induced changes to cell morphology, the localization of subcellular structures, intra-cellular diffusion (e.g., NCT), and the dynamic responses of key molecules of interest, to name a few.

The rise of nanoscience and nanotechnology would not have been happened without the impressive development of instruments that allow to resolve structure on the nanometer scale with atomic resolution. Examples are scanning-probe and electron microscopy techniques. In recent years, several major breakthroughs gave rise to an exceptional boost in the performance of today's electron microscopy (EM), both for solid-state and soft (e.g. biological) materials: 1) high-resolution through image corrections, 2) fast and highly efficient electron detectors, 3) efficient artifact-free sample fabrication (cryo-EM and FIBEM), and 4) 3D tomography and image reconstruction. This has given a leap to what can be imaged today, allowing for example to reconstruct the atomic structure of single proteins and image complex interfaces in solid-state materials with atomic scale. The University of Basel (UBAS) is nationally and internationally recognized as a leader in nanoscience and nanotechnology. It was the leading house of the National Center in Competence and Research (NCCR) on Nanoscience, which later became the Swiss Nanoscience Institute (SNI), the institution that submits the current proposal. UBAS is also co-leading the NCCR Molecular Systems Engineering and the NCCR QSIT on Quantum Science (both together with ETHZ). Nanoscience is a focus area in the research portfolio of UBAS and instrumental for the recent development of quantum science. The present proposal to the SNF R'Equip scheme has been put together by key researchers at UBAS who work on current topics in nanoscience and nanotechnology in various disciplines from quantum science, material science, polymer chemistry to molecular biology, and, who make use of EM available within the SNI. The principle investigators, who submit this proposal together, do research that relies on the availability of state-of-the-art nanoimaging tools, such as a transmission electron microscope (TEM). The proposal outlines a convincing case for the purchase of special, unique TEM that combines state-of-the-art (and fast) atomic resolution imaging with material analysis using EDX and scanning TEM (STEM). This combination is unique and crucial for the University of Basel to stay at the forefront of science.

Molecular Systems Engineering is a National Centre of Competence in Research (NCCR) funded by the Swiss National Science Foundation (SNSF), and headed by the University of Basel and the ETH Zurich. This NCCR combines expertise from chemistry, biology, physics, bioinformatics, and engineering. The overreaching aim is to develop tools and devices to monitor and manipulate off-equilibrium (bio)chemical systems. These may find applications in the synthesis of high added-value products, as innovative diagnostic tools and for the restoration of a desired cellular or organ function.

The goal of this project is to fully optimize AFM and optical nanoscopy measurements of live cells, and thereby be able to resolve subcellular structures and their dynamic responses to external force. Our long-term goal is to correlate cellular mechanics to the interplay between the microenvironment, nucleo/cytoarchitecture, and the protein linkages between them.

This project seeks to integrate atomic force microscopy (AFM) with spinning disk confocal microscopy including fluorescence recovery after photobleaching and a laser ablation system. This so-called "mechano-optical microscope" (MOM) will fully optimize AFM and optical measurements of live cells, and thereby be able to resolve subcellular structures and their dynamic responses to external force. Our long term goal is to construct a multi-scale rheological model 4 that correlates cellular mechanics to the interplay between the microenvironment, nucleo/cytoarchitecture, and the protein linkages between them.