Nucleocytoplasmic traffic is one of the hallmarks of the eukaryotic cell. To a large extent, this process is mediated by nuclear import and export receptors. Study of nuclear import and export has... Show moreNucleocytoplasmic traffic is one of the hallmarks of the eukaryotic cell. To a large extent, this process is mediated by nuclear import and export receptors. Study of nuclear import and export has had wide implications for many cellular processes including basic cell metabolism, cellular differentiation and disease. In this thesis I have studied the nuclear export receptor CRM1/exportin1 and its interaction with various transport substrates and the nuclear pore complex (NPC), the supramolecular structure through which receptor-mediated nucleocytoplasmic transport takes place. CRM1 interacts with its substrate through the nuclear export signal (NES) and I have identified a novel type NES, the supraphysiological NES (supraNES). This is a synthetic peptide NES that binds CRM1 with an unusual high affinity. Interestingly, we found that supraNESs do not only exist as synthetic peptides, but are present in certain viruses, where they are required for export of the viral particle from the nucleus. The currently emerging supraNESs in other viruses and the hypothesis that supraNESs could be responsible for export of large cargo in general will be discussed. Show less
Antigen presentation by MHC class II is critical for immune responses against pathogens and tumors. Antigen loading occurs primarily in lysosomal-related organelles (LROs) known as MIICs.... Show moreAntigen presentation by MHC class II is critical for immune responses against pathogens and tumors. Antigen loading occurs primarily in lysosomal-related organelles (LROs) known as MIICs. Ultimately, the MHC II-peptide complexes are transported for cell surface display. Here, we study intracellular transport of MIICs, a poorly understood process in MHC II antigen presentation. We propose that Rab7 lies at the heart of transport by assembling a specific receptor (Rab7-RILP-ORP1L) for the minus end-directed dynein-dynactin motor on the cytosolic face of MIICs/LROs. Full activation of transport requires a second receptor, _III spectrin. Whereas this model explains how Rab7 controls minus end-directed transport, it does not suffice to explain the characteristic pattern of bidirectional motility exhibited by MIICs/LROs. Here, we propose that cholesterol dictates ORP1L conformation which acts as a switch controlling access of dynein-dynactin to Rab7-RILP, thereby regulating LRO positioning, as observed in NPC disease. Rab7-RILP-ORP1L may also integrate transport and tethering of MIICs/LROs, consecutive processes within the endocytic pathway. Show less
_-Catenin is an important protein for cancer research as it influences numerous events in the cell that lead to the development of cancer when gone awry. At the adherens junctions, _-catenin... Show more_-Catenin is an important protein for cancer research as it influences numerous events in the cell that lead to the development of cancer when gone awry. At the adherens junctions, _-catenin functions in cell-cell adhesion to maintain epithelial organisation. As an effector of Wnt signaling, _-catenin controls numerous developmental processes as well as homeostatic self-renewal. The effector function of _-catenin is to form a transcriptional complex in the nucleus with TCF/Lef transcription factors to regulate target gene expression. Due to the dual function of _-catenin in cell adhesion and signaling, there are different pools of the protein. The research described in this thesis focuses on the role of _-catenin in the Wnt signaling pathway. What is the pool of _-catenin that is active in signaling? Where is active _-catenin localized? Where and how is _-catenin activated and how is its nuclear export regulated to terminate Wnt signaling. Chapter 1 provides a general introduction about the different aspects of nuclear transport and the Wnt signaling cascade, putting it into the context of cancer development. Chapter 2 describes the identification of Ran-binding protein 3 (RanBP3) as a novel regulator of the active signaling form of _-catenin. We initiated this study to investigate the nuclear translocation of _-catenin and found that RanBP3 directly inhibits _-catenin signaling by stimulating nuclear export of the transcriptionally active form of _-catenin. The active form of _-catenin is unphosphorylated on its N-terminus, and covers only a small fraction of the total amount of _-catenin in the cell. We therefore continued to study the localization of this pool of _-catenin in Chapter 3. We describe that a relative large pool of unphosphorylated _-catenin resides at the adherens junctions, where it most likely functions in cell-cell adhesion. As Wnt treatment induces recruitment of unphosphorylated _-catenin to the plasma membrane, it is impossible to distinguish the resident junctional pool of unphosphorylated _-catenin from the signaling pool. We emphasize the importance of an E-cadherin null background in studying signaling competent unphosphorylated _-catenin. In Chapter 4, we study the unphosphorylated _-catenin pool at the plasma membrane upon Wnt signal induction in E-cadherin knock out cells. Plasma membrane recruitment of _-catenin in the early steps of the Wnt signaling cascade fits with recent new insights, which suggest recruitment of Axin and Dvl to the activated Wnt receptor LRP5/6. We expand these insights by showing that active _-catenin, Axin, APC and activated LRP6 receptor all localize to the plasma membrane upon Wnt stimulation. Moreover, we find that Wnt induced _-catenin is transcriptionally more active than overexpressed _-catenin. We suggest a model in which plasma membrane recruitment of _-catenin represents an important step in _-catenin processing and Wnt signal transduction. In Chapter 5, we determine the nuclear export kinetics of _-catenin in human cells and show that _-catenin exits the nucleus very fast, independently of the CRM1 export pathway and that _-catenin can enhance export of the small molecule GFP (green fluorescent protein). These observations fit into a model in which _-catenin can translocate quickly into and out of the nucleus independently of nuclear transport receptors. Therefore, the activity and localization of _-catenin are likely to be regulated by retention of the protein in the nucleus, cytoplasm and plasma membrane. Finally, in Chapter 6 we reconcile our findings with current knowledge of the Wnt signaling cascade. Show less
