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Abstract

Transport of molecules between the nucleus and the cytoplasm is tightly regulated by the nuclear pore complex (NPC), the "gatekeeper" of the nucleus. NPCs allow passage of small molecules (< 5 nm), whereas larger cargoes can only cross the permeability barrier if they are bound to nuclear transport receptors (NTRs), which recognize specific nuclear import and export signals (NLSs and NESs) on their surface. Among the thousands of molecules that travel through the NPC, an important class is represented by large cargoes (> 15 nm), such as pathogens, mRNAs and pre-ribosomal subunits. Strikingly, it has been observed that NPCs can accommodate Hepatitis B virus capsids of 36 nm in diameter, almost comparable with the dimensions of the central channel itself. The mechanism for the transport of cargoes with such exceptional size are still unclear, especially the role of multivalent NTR binding when several NLSs are present on the cargo surface. In my PhD thesis I characterized the nuclear import properties of large cargoes in cells. In order to do so, I firstly developed a large cargo model suitable for nuclear transport studies based on virus-like particles. It comprises more than 20 different cargoes with size range 18-36 nm and different amount of NLSs exposed on their surface. Following the development of a semi-automatic kinetic cellular transport assay, I have shown that large cargoes require a minimum number of bound NTRs in order to achieve productive entry into the nucleus, and I further found evidence of a linear relationship between #NLSs and import efficiency. Moreover, the experiments revealed that the minimum threshold for import increases non-linearly with size, reaching up to an unprecedented 100 NTRs per cargo and pointing to a high drop in the transport efficiency for cargoes > 25 nm, which could represent a previously undescribed “second gate” at the NPC. In order to go beyond the global picture from bulk import kinetics, I developed a microscopy setup and LabVIEW software for high-resolution studies of large cargo import in cells. Performing dual-color super-resolution imaging of nucleoporins and cargoes, I have shown that HBV capsids specifically dock on NPCs and interact with both the cytoplasmic component and directly with the permeability barrier. Additional single-particle tracking experiments allow me now to follow the import process with high spatiotemporal resolution, dissecting the multiple steps involved in large cargo import (docking, barrier crossing, release into the nucleus). Integrating this knowledge with the findings from bulk kinetics studies and super-resolution microscopy paves the way for an integrated view of large cargo transport through the NPC.