Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes

Breakdown of the nuclear envelope (NE) was analyzed in live starfish oocytes using a size series of fluorescently labeled dextrans, membrane dyes, and GFP-tagged proteins of the nuclear pore complex (NPC) and the nuclear lamina. Permeabilization of the nucleus occurred in two sequential phases. In phase I the NE became increasingly permeable for molecules up to ∼40 nm in diameter, concurrent with a loss of peripheral nuclear pore components over a time course of 10 min. The NE remained intact on the ultrastructural level during this time. In phase II the NE was completely permeabilized within 35 s. This rapid permeabilization spread as a wave from one epicenter on the animal half across the nuclear surface and allowed free diffusion of particles up to ∼100 nm in diameter into the nucleus. While the lamina and nuclear membranes appeared intact at the light microscopic level, a fenestration of the NE was clearly visible by electron microscopy in phase II. We conclude that NE breakdown in starfish oocytes is triggered by slow sequential disassembly of the NPCs followed by a rapidly spreading fenestration of the NE caused by the removal of nuclear pores from nuclear membranes still attached to the lamina

Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells

During mitosis 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 present here our quantitative assay based on confocal time-lapse microscopy of single dividing cells. We use this assay 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. Our data establish that NPC assembly is an ordered stepwise process that leads to import function already in a partially assembled state. We furthermore find that nucleoporin dissociation does not occur in the reverse order from binding during assembly, which may indicate a distinct mechanism

Dynamique de l'organisation des pores nucléaires

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Function and regulation of protein neddylation. ‘Protein Modifications: Beyond the Usual Suspects' Review Series

Neddylation is the post-translational protein modification that is most closely related to ubiquitination. However, ubiquitination is known to regulate a myriad of processes in eukaryotic cells, whereas only a limited number of neddylation substrates have been described to date. Here, we review the principles of protein neddylation and highlight the mechanisms that ensure the specificity of neddylation over ubiquitination. As numerous neddylation substrates probably remain to be discovered, we propose some criteria that could be used as guidelines for the characterization of neddylated proteins

Characterization of the cellular network of ubiquitin conjugating and ligating enzymes

International audienc

Fluorescence perturbation techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, photoactivation, photoconversion, and FLIP.

International audienceThe technique of fluorescence recovery after photobleaching (FRAP) was introduced in the mid-1970s to study the diffusion of biomolecules in living cells. For several years, it was used mainly by a small number of biophysicists who had developed their own photobleaching systems. Since the mid-1990s, FRAP has gained increasing popularity because of the conjunction of two factors: First, photobleaching techniques are easily implemented on confocal laser-scanning microscopes (CLSMs), and so FRAP has become available to anyone who has access to such equipment. Second, the advent of green fluorescent protein (GFP) has allowed easy fluorescent tagging of proteins and their observation in living cells. Thanks both to the versatility of modern CLSMs, which allow control of laser intensity at any point of the image, and to the development of new fluorescent probes, additional photoperturbation techniques have emerged during the last few years. After the photoperturbation event, one observes and then analyzes how the fluorescence distribution relaxes toward the steady state. Because the photochemical perturbation of suitable fluorophores is essentially irreversible, changes of fluorescence intensity in the perturbed and unperturbed regions are due to the exchange of tagged molecules between those regions. This article first discusses the materials required for performing FRAP experiments on a CLSM and the software for data analysis. It then describes general considerations on how to perform FRAP experiments as well as the necessary controls. Finally, different possible ways to analyze the data are presented

Expression of EGFP-amino-tagged human mu opioid receptor in Drosophila Schneider 2 cells: a potential expression system for large-scale production of G-protein coupled receptors.

The G-protein coupled receptor (GPCR) human mu opioid receptor (hMOR) fused to the carboxy-terminus of the enhanced green fluorescent protein (EGFP) has been successfully and stably expressed in Drosophila Schneider 2 cells under the control of an inducible metallothionein promoter. Polyclonal cells expressing EGFPhMOR display high-affinity, saturable, and specific binding sites for the opioid antagonist diprenorphine. Competition studies with opioid agonists and antagonists defined the pharmacological profile of a mu opioid receptor similar to that observed in mammalian cells, suggesting proper folding of EGFPhMOR in a high-affinity state in Drosophila cells. The functionality of the fusion protein was demonstrated by the ability of agonist to reduce forskolin-stimulated cyclic AMP production and to induce [35S]GTPgammaS incorporation. The EGFPhMOR protein had the expected molecular weight (70kDa), as demonstrated by protein immunoblotting with anti-EGFP and anti-C-terminus hMOR antibodies. However, quantitative EGFP fluorescence intensity analysis revealed that the total level of expressed EGFPhMOR is 8-fold higher than the level of diprenorphine binding sites, indicating that part of the receptor is not in a high-affinity state. This may in part be due to a population of receptors localized in intracellular compartments, as shown by the distribution of fluorescence between the plasma membrane and the cell interior. This study shows that EGFP is a valuable and versatile tool for monitoring and quantifying expression levels as well as for optimizing and characterizing an expression system. Optimization of the Drosophila Schneider 2 cell expression system will allow large-scale purification of GPCRs, thus enabling structural studies to be undertaken

The Entire Nup107-160 Complex, Including Three New Members, Is Targeted as One Entity to Kinetochores in Mitosis

In eukaryotes, bidirectional transport of macromolecules between the cytoplasm and the nucleus occurs through elaborate supramolecular structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs). NPCs are composed of multiple copies of ∼30 different proteins termed nucleoporins, of which several can be biochemically isolated as subcomplexes. One such building block of the NPC, termed the Nup107-160 complex in vertebrates, was so far demonstrated to be composed of six different nucleoporins. Here, we identify three WD (Trp-Asp)-repeat nucleoporins as new members of this complex, two of which, Nup37 and Nup43, are specific to higher eukaryotes. The third new member Seh1 is more loosely associated with the Nup107-160 complex biochemically, but its depletion by RNA interference leads to phenotypes similar to knock down of other constituents of this complex. By combining green fluorescent protein-tagged nucleoporins and specific antibodies, we show that all the constituents of this complex, including Nup37, Nup43, Seh1, and Sec13, are targeted to kinetochores from prophase to anaphase of mitosis. Together, our results indicate that the entire Nup107-160 complex, which comprises nearly one-third of the so-far identified nucleoporins, specifically localizes to kinetochores in mitosis

RanBP2/Nup358 Provides a Major Binding Site for NXF1-p15 Dimers at the Nuclear Pore Complex and Functions in Nuclear mRNA Export

Metazoan NXF1-p15 heterodimers promote the nuclear export of bulk mRNA across nuclear pore complexes (NPCs). In vitro, NXF1-p15 forms a stable complex with the nucleoporin RanBP2/Nup358, a component of the cytoplasmic filaments of the NPC, suggesting a role for this nucleoporin in mRNA export. We show that depletion of RanBP2 from Drosophila cells inhibits proliferation and mRNA export. Concomitantly, the localization of NXF1 at the NPC is strongly reduced and a significant fraction of this normally nuclear protein is detected in the cytoplasm. Under the same conditions, the steady-state subcellular localization of other nuclear or cytoplasmic proteins and CRM1-mediated protein export are not detectably affected, indicating that the release of NXF1 into the cytoplasm and the inhibition of mRNA export are not due to a general defect in NPC function. The specific role of RanBP2 in the recruitment of NXF1 to the NPC is highlighted by the observation that depletion of CAN/Nup214 also inhibits cell proliferation and mRNA export but does not affect NXF1 localization. Our results indicate that RanBP2 provides a major binding site for NXF1 at the cytoplasmic filaments of the NPC, thereby restricting its diffusion in the cytoplasm after NPC translocation. In RanBP2-depleted cells, NXF1 diffuses freely through the cytoplasm. Consequently, the nuclear levels of the protein decrease and export of bulk mRNA is impaired

Rtt101 and Mms1 in budding yeast form a CUL4DDB1-like ubiquitin ligase that promotes replication through damaged DNA

In budding yeast the cullin Rtt101 promotes replication fork progression through natural pause sites and areas of DNA damage, but its relevant subunits and molecular mechanism remain poorly understood. Here, we show that in budding yeast Mms1 and Mms22 are functional subunits of an Rtt101-based ubiquitin ligase that associates with the conjugating-enzyme Cdc34. Replication forks in mms1Δ, mms22Δ and rtt101Δ cells are sensitive to collisions with drug-induced DNA lesions, but not to transient pausing induced by nucleotide depletion. Interaction studies and sequence analysis have shown that Mms1 resembles human DDB1, suggesting that Rtt101Mms1 is the budding yeast counterpart of the mammalian CUL4DDB1 ubiquitin ligase family. Rtt101 interacts in an Mms1-dependent manner with the putative substrate-specific adaptors Mms22 and Crt10, the latter being a regulator of expression of ribonucleotide reductase. Taken together, our data suggest that the Rtt101Mms1 ubiquitin ligase complex might be required to reorganize replication forks that encounter DNA lesions