Elucidating the mechanism of nuclear pore complex assembly in intact nuclei of live cells
Zusammenfassung der Projektergebnisse
2.3. Summary of achievements In higher eukaryotes, nuclear pore complexes (NPCs) disassemble in prophase and are rebuilt in anaphase and telophase. NPC formation is hypothesized to occur by the interaction of mitotically stable subcomplexes that form defined structural intermediates. To determine the sequence of events that lead to breakdown and reformation of functional NPCs during mitosis we developed a quantitative assay based on confocal time-lapse microscopy of single dividing cells in order to systematically investigate the kinetics of dis- and reassembly for eight nucleoporin subcomplexes relative to nuclear transport in NRK cells, linking the assembly state of the NPC with its function (Dultz et al., 2008). Our data established that NPC assembly is an ordered stepwise process. However, we found that nucleoporin dissociation does not occur in the reverse order from binding during assembly, which may indicate a distinct mechanism. Furthermore, we found that the nuclear import function of the NPC is reestablished within <10 min after anaphase onset, despite the fact that the pore is not yet fully assembled. Since Abschlussbericht DFG EL 246/3-1+2 NPC assembly in intact nuclei 4 it is widely assumed that import functionality simultaneously leads to the exclusion of bulk cytoplasmic proteins, the fact that the NPC assembly is not fully completed when import capacity is regained raised the question of whether the transport and permeability barrier functions of the nuclear envelope are indeed coupled. To address this question, we analyzed the reestablishment of the permeability barrier of the nuclear envelope after mitosis in living cells by monitoring the flux of the reversibly photoswitchable fluorescent protein Dronpa from the cytoplasm into the nucleus after photoactivation (Dultz et al., 2009). We performed many consecutive flux measurements in the same cell to directly monitor changes in nuclear envelope permeability. Our measurements at different time points after mitosis in individual cells showed that contrary to the general view and despite the rapid reestablishment of facilitated nuclear import, the nuclear envelope remains relatively permeable for passive diffusion for the first 2 h after mitosis. Our data demonstrated that reformation of the permeability barrier of nuclear pore complexes occurs only gradually and is uncoupled from regaining active import functionality. In metazoa, new NPCs form at two different cell cycle stages: at the end of mitosis and during interphase. Since the mechanisms of these two assembly processes may differ, we next focused on the dynamics of interphase assembly. To this end we applied high-resolution live cell microscopy to analyze the dynamics of single NPCs in living mammalian cells during interphase (Dultz et al., 2010). We showed that nuclear growth and NPC assembly are correlated and occur at a constant rate throughout interphase. By analyzing the kinetics of individual NPC assembly events, we demonstrated that they are initiated by slow accumulation of the membrane nucleoporin Pom121 followed by the more rapid association of the soluble NPC subcomplex Nup107-160, that is in an inverse order of recruitment to the post-mitotic assembly. Additionally, the overall much slower kinetics compared with postmitotic NPC assembly supports the conclusion that the two processes occur by distinct molecular mechanisms. The live cell imaging methodologies we developed in the framework of the project, also allowed us to contributed to the study of the NPC-assembly independent role of nucleoporin Nup133 in an international collaboration. Together with the group of Valerie Doye at the Institut Monod in Paris, we demonstrated that the N-terminal domain of Nup133 is required for efficient anchoring of the dynein/dynactin complex to the NE in prophase (Bolhy et al., 2011). Nup133 exerts this function through an interaction network via the kinetochore components CENP-F and NudE/EL. We showed that this molecular chain is critical for maintaining centrosome association with the NE at mitotic entry and contributes to this process without interfering with the previously described RanBP2-BICD2-dependent pathway of centrosome anchoring. Finally, the study revealed that tethering of centrosomes to the Abschlussbericht DFG EL 246/3-1+2 NPC assembly in intact nuclei 5 nuclear surface at the G2/M transition, along with other cellular mechanisms, contributes, to early stages of bipolar spindle assembly. While our results in the framework of this projects have provided the first kinetic molecular kinetic analysis of both postmitotic and interphase NPC assembly, a true mechanistic understanding of this process will also require to know the structural organization of the assembly intermediates with molecular specificity. The NPC is a prominent example of an essential molecular machine, whose structural organization remains undetermined, due to its size and complexity. Although atomic structures of in vitro purified proteins and small complexes can be obtained by x-ray crystallography or NMR, methods applicable to large complexes in situ, e.g., electron tomography, currently lack the resolution for direct molecular assignments. To address this methodological gap, we therefore combined stochastic superresolution (SR) microscopy, to directly resolve the ring-like structure of the NPC, with single particle averaging, to use information from thousands of pores to increase the resolution to the molecular scale. Using this novel approach we could recently determine the position of fluorescent molecular labels in the NPC with a precision well below 1 nm, an order of magnitude higher than previously possible by superresolution microscopy (Szymborska et al., submitted). Applying this approach systematically to the largest building block of the NPC, the Nup107-160 subcomplex, we obtained new insight into the structure of the NPC scaffold. Our method can be used to study the molecular organization of many large protein complexes in situ in whole cells and it systematic application in the future will allow us to dissect the structural intermediates of NPC assembly intermediates both structurally and molecularly.
Projektbezogene Publikationen (Auswahl)
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2008. Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells. J Cell Biol. 180:857-65
Dultz, E., E. Zanin, C. Wurzenberger, M. Braun, G. Rabut, L. Sironi, and J. Ellenberg
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2009. Formation of the nuclear envelope permeability barrier studied by sequential photoswitching and flux analysis. Biophys J. 97:1891-7
Dultz, E., S. Huet, and J. Ellenberg
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2010. Live imaging of single nuclear pores reveals unique assembly kinetics and mechanism in interphase. J Cell Biol. 191:15-22
Dultz, E., and J. Ellenberg
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2011. A Nup133-dependent NPC-anchored network tethers centrosomes to the nuclear envelope in prophase. J Cell Biol. 192:855-71
Bolhy, S., I. Bouhlel, E. Dultz, T. Nayak, M. Zuccolo, X. Gatti, R. Vallee, J. Ellenberg, and V. Doye
