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 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.

The atomic force microscope (AFM) has emerged as a powerful tool to quantify cellular nanomechanics at the cellular and molecular level. Nevertheless, in the context of in vivo cellular studies, AFM is limited in terms of resolving (i) the biochemical identity of biological structures, (ii) subcellular structures, and (iii) their dynamic responses to external force. To circumvent these limitations, AFM is often combined with fluorescence microscopy to image cellular shape and labeled cellular proteins while making force measurements. However, this typically provides "in-plane" (XY) views of the sample parallel to the surface plane. Yet, the most significant cellular deformations and cytoskeletal rearrangements are aligned perpendicular to the surface plane (XZ). Hence, specific sub-cellular conformational changes along the loading direction can be directly correlated to the applied AFM load by fluorescence imaging in the XZ plane. This will enable us to dissect and assign the mechanical contributions of the intra- and inter-cellular components to mechano-phenotypes of living cells.

Our motivation is to overcome key shortcomings of current GPCR lab-on-a-chip nanoscale drug discovery and extend the established and well accepted but limited SPR-based drug screening by new possibilities to address inherently difficult-to-screen GPCRs, and more than that, to obtain detailed functional information about differences of ligands at nanoscale and the biological effects that they trigger for more comprehensive drug profiling. In fact, the need to identify drugs that discriminate between G protein and arrestin signaling has revolutionized GPCR drug discovery setting paradigm shifts and defining "biased agonism" or "functional selectivity". Investment-intense activities can be observed in terms of patent disclosures for new chemical entities with such properties by Biotech, but also large Pharma companies. NanoGhip finds application not only in lab-on-a-chip nanoscale drug screening, but also assists struc-ture-based drug discovery (SBDD), e.g. for determination of ideal crystallization conditions. Of course, NanoGhip could be as well implemented in diagnostic lab-on-a-chip tools to detected overregulated na-tive ligands as indicators of disease on-site.