The vertebrate nuclear pore complex, 30 times the size of a ribosome, assembles from a library of soluble subunits and two membrane proteins. Using immunodepletion of Xenopus nuclear reconstitution extracts, it has previously been possible to assemble nuclei lacking pore subunits tied to protein import, export, or mRNA export. However, these altered pores all still possessed the bulk of pore structure. Here, we immunodeplete a single subunit, the Nup107-160 complex, using antibodies to Nup85 and Nup133, two of its components. The resulting reconstituted nuclei are severely defective for NLS import and DNA replication. Strikingly, they show a profound defect for every tested nucleoporin. Even the integral membrane proteins POM121 and gp210 are absent or unorganized. Scanning electron microscopy reveals pore-free nuclei, while addback of the Nup107-160 complex restores functional pores. We conclude that the Nup107-160 complex is a pivotal determinant for vertebrate nuclear pore complex assembly.

Assembly of a eukaryotic nucleus involves three distinct events: membrane recruitment, fusion to form a double nuclear membrane, and nuclear pore complex (NPC) assembly. We report that importin ␤ negatively regulates two of these events, membrane fusion and NPC assembly. When excess importin ␤ is added to a full Xenopus nuclear reconstitution reaction, vesicles are recruited to chromatin but their fusion is blocked. The importin ␤ down-regulation of membrane fusion is Ran-GTP reversible. Indeed, excess RanGTP (RanQ69L) alone stimulates excessive membrane fusion, leading to intranuclear membrane tubules and cytoplasmic annulate lamellae-like structures. We propose that a precise balance of importin ␤ to Ran is required to create a correct double nuclear membrane and simultaneously to repress undesirable fusion events. Interestingly, truncated importin ␤ 45-462 allows membrane fusion but produces nuclei lacking any NPCs. This reveals distinct importin ␤-regulation of NPC assembly. Excess full-length importin ␤ and ␤ 45-462 act similarly when added to prefused nuclear intermediates, i.e., both block NPC assembly. The importin ␤ NPC block, which maps downstream of GTP␥S and BAPTA-sensitive steps in NPC assembly, is reversible by cytosol. Remarkably, it is not reversible by 25 M RanGTP, a concentration that easily reverses fusion inhibition. This report, using a full reconstitution system and natural chromatin substrates, significantly expands the repertoire of importin ␤. Its roles now encompass negative regulation of two of the major events of nuclear assembly: membrane fusion and NPC assembly. INTRODUCTIONIn cells from yeast to mammals, importin ␣ and ␤ act together to ferry classical nuclear localization signal (NLS)-bearing proteins into the nucleus (Gorlich and Kutay, 1999;Stoffler et al., 1999;Damelin and Silver, 2000;Rout et al., 2000;Conti and Izaurralde, 2001;Vasu and Forbes, 2001;Damelin et al., 2002;Weis, 2003). Once in the nucleus the small GTPase Ran binds to importin ␤, displacing importin ␣ and the NLS cargo, thus completing import. In the nucleus, Ran is kept in a GTP state by the constant action of its chromatinbound Ran-GEF, RCC1 (Melchior and Gerace, 1998;Macara, 2001;Dasso, 2002;Kalab et al., 2002;Schwoebel et al., 2002;Steggerda and Paschal, 2002). In contrast, RanGDP is the predominant form found in the cytoplasm due to the cytoplasmic localization of RanGAP.The horizons for importin ␣ and ␤ were unexpectedly broadened when they were found to play a very different role at mitosis. In metazoans, importin ␣ and ␤ are released to the cytosol by nuclear breakdown, where they act to inhibit proteins essential for mitotic spindle assembly. However, the inhibition of spindle assembly is reversed by Ran in the vicinity of mitotic chromosomes, where RanGTP continues to be produced by chromatin-bound RCC1 (Kalab et al., 1999;Gruss et al., 2001;Nachury et al., 2001;Wiese et al., 2001;Dasso, 2002). Thus, a spindle forms only around the mitotic (ER) chromosomes and not elsewhere in the cytoplasm.At the end ...

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca(2+)-calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca(2+)-calmodulin or Ca(2+)-S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.

and Michael Elbaum, michael.elbaum@weizmann.ac.il † These authors contributed equally to this work.Delivery of DNA to the cell nucleus is an essential step in many types of viral infection, transfection, gene transfer by the plant pathogen Agrobacterium tumefaciens and in strategies for gene therapy. Thus, the mechanism by which DNA crosses the nuclear pore complex (NPC) is of great interest. Using nuclei reconstituted in vitro in Xenopus egg extracts, we previously studied DNA passage through the nuclear pores using a single-molecule approach based on optical tweezers. Fluorescently labeled DNA molecules were also seen to accumulate within nuclei. Here we find that this import of DNA relies on a soluble protein receptor of the importin family. To identify this receptor, we used different pathway-specific cargoes in competition studies as well as pathway-specific dominant negative inhibitors derived from the nucleoporin Nup153. We found that inhibition of the receptor transportin suppresses DNA import. In contrast, inhibition of importin β has little effect on the nuclear accumulation of DNA. The dependence on transportin was fully confirmed in assays using permeabilized HeLa cells and a mammalian cell extract. We conclude that the nuclear import of DNA observed in these different vertebrate systems is largely mediated by the receptor transportin. We further report that histones, a known cargo of transportin, can act as an adaptor for the binding of transportin to DNA. Many viruses have developed specific strategies to promote the nuclear uptake of their genomes by the host (1,2). The addition of exogenous oligonucleotides to cells has separately been used for both viral-and nonviral-based gene therapy (3,4). In some studies, genetic transformation by the added DNA can take place on a time scale faster than cell division, implying that DNA import across the interphase nuclear envelope must occur in vivo (5). Indeed, a large number of studies have gone on to demonstrate that DNA entry through the nuclear pore occurs and can be enhanced by artificially coupling the DNA to peptides containing nuclear localization signals (NLSs), implying translocation across the nuclear pore (6-14).

A method for creating microscale‐patterned surfaces by direct‐write lithography is described. A tightly focused, low‐power infrared laser beam is applied to a homogeneous precursor solution containing soluble reagents. When the laser is focused directly at a glass–solution interface, it initiates the local precipitation of a solid product that attaches firmly to the substrate. Operating the laser momentarily forms isolated spots, whereas moving the microscope stage or the laser spot draws continuous lines. The method has been demonstrated for metallic silver and gold, for oxidized copper, and for molybdenum disulfide, suggesting a broad range of suitable materials. Silver patterns were further modified by chemical reactions. Their morphology and physical properties can be altered during deposition by the use of capping agents, which may provide an onset for further functionalization.