Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death. Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in ageing mice. In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases (HAT), suppressing oxidative stress and necrosis. Conversely, depletion of endogenous polyamines led to hyperacetylation, generation of reactive oxygen species, early necrotic death and decreased lifespan. The altered acetylation status of the chromatin led to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, worms and human cells. Finally, we found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity.

The nuclear pore complex regulates cargo transport between the cytoplasm and the nucleus. We set out to correlate the governing biochemical interactions to the nanoscopic responses of the phenylalanineglycine (FG)-rich nucleoporin domains, which are involved in attenuating or promoting cargo translocation. We found that binding interactions with the transport receptor karyopherin-beta1 caused the FG domains of the human nucleoporin Nup153 to collapse into compact molecular conformations. This effect was reversed by the action of Ran guanosine triphosphate, which returned the FG domains into a polymer brush-like, entropic barrier conformation. Similar effects were observed in Xenopus oocyte nuclei in situ. Thus, the reversible collapse of the FG domains may play an important role in regulating nucleocytoplasmic transport.

Over the past two years, it has become evident that there is an unexpected link between nuclear pore complex structure and dynamics, nucleocytoplasmic transport and chromosome segregation. In addition, a tomographic three-dimensional reconstruction of native nuclear pore complexes preserved in thick amorphous ice has unveiled a number of new structural features of this supramolecular machine. These data, together with some of the elementary physical principles that underlie nucleocytoplasmic transport, will be discussed in this review.

We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.Transport through nuclear pores requires concerted action between the structural components of the nuclear pore complex (NPC) and the soluble transport factors that bind to the transport substrates and shuttle between the nuclear and cytoplasmic compartments (for reviews, see references 2 and 31). Substantial progress toward an understanding of nuclear protein import has been achieved in the past few years, but very little is known about how RNA is exported from the nucleus into the cytoplasm. Among the factors required for nuclear protein import are the classical nuclear localization signalreceptor complex, consisting of importin/karyopherin ␣ and ␤, the small GTPase Ran, and several Ran-binding proteins, as well as repeat sequences containing nucleoporins (for reviews, see references 5 and 11). Recently, additional routes of import into the nucleus were discovered, suggesting that major classes of transport substrates use different import pathways. Transportin and Kap123p were identified as novel transport factors that bind directly to their transport substrates, hnRNP protein A1 and ribosomal protein L25, respectively (30, 38). Transportin and Kap123p belong to a growing family of importin ␤-like proteins which have a Ran GTP-binding domain in their amino-terminal portions (5, 10). Recently, Mtr10p, which is also a member of this protein family, was shown to be the importin for yeast Np13p (34,41). An essential role for Ran in energydependent nuclear protein import has been firmly established, but how Ran and the many Ran activity-modulating proteins participate in the actual translocation process is still controversial.The Ran system is also involved in transport from the nucleus (9,14,18,36). It has been firmly established that nuclear export sequences (NES), first identified in viral proteins such as human immunodeficiency virus Rev and protein kinase inhibitor, mediate the exit of proteins from the nucleus (for a review, see reference 8). For the Rev protein, which is an RNA-binding protein with a specificity for unspliced or partially spliced viral transcripts, viral mRNA is coexported through association with Rev (3). Initially, it was found that Rev NES interact with Rip (6,47), which resembles repeat sequence-containing nucleoporins and accordingly was suggested to be a NES receptor at the nuclear pores. Recen...

Natively unfolded phenylalanine-glycine (FG)-repeat domains are alleged to form the physical constituents of the selective barriergate in nuclear pore complexes during nucleocytoplasmic transport. Presently, the biophysical mechanism behind the selective gate remains speculative because of a lack of information regarding the nanomechanical properties of the FG domains. In this work, we have applied the atomic force microscope to measure the mechanical response of individual and clusters of FG molecules. Single-molecule force spectroscopy reveals that FG molecules are unfolded and highly flexible. To provide insight into the selective gating mechanism, an experimental platform has been constructed to study the collective behavior of surface-tethered FG molecules at the nanoscale. Measurements indicate that the collective behavior of such FG molecules gives rise to an exponentially decaying long-range steric repulsive force. This finding indicates that the molecules are thermally mobile in an extended polymer brush-like conformation. This assertion is confirmed by observing that the brush-like conformation undergoes a reversible collapse transition in less polar solvent conditions. These findings reveal how FGrepeat domains may simultaneously function as an entropic barrier and a selective trap in the near-field interaction zone of nuclear pore complexes; i.e., selective gate.force spectroscopy ͉ nanomechanics ͉ natively unfolded proteins ͉ nuclear pore complex ͉ selective gating N ucleocytoplasmic transport describes the exchange of molecular cargo between the nucleus and the cytoplasm across numerous perforations in the nuclear envelope called nuclear pore complexes (NPCs) (1). Each vertebrate NPC is an Ϸ120-MDa supramolecular complex consisting of Ϸ30 different proteins called nucleoporins (or Nups) that form an eightfold symmetric central framework embracing a central pore. The cross-section of the central pore reveals an hourglass-like channel that is Ϸ90 nm long and is narrowest (diameter of Ϸ40 nm) at the NPC's midplane (1). Whereas small molecules such as water and ions proceed freely by passive diffusion (2), the NPC poses a barrier to larger molecular cargo (Ͼ20 kDa) that do not harbor nuclear localization signals (NLSs) (3). Conversely, the barrier does not seem to hinder the passage of NLS cargo when in complex with a transport receptor (e.g., Karyopherin͞Importin) (4). Moreover, because receptormediated transport is rapid even for large NLS cargoes (5), it is apparent that the NPC-selective gating mechanism is not solely based on size exclusion.Presently, an unambiguous understanding of the gating mechanism remains elusive because of a lack of information regarding the mechanical aspects of the molecular components that make up the NPC. Emerging evidence indicates that gating is closely correlated with the interactions and spatial organization of nucleoporins containing phenylalanine-glycine (FG)-repeat domains (called FG domains) (1). Importantly, FG domains exhibit low overall hydrophobicity and are...

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The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.