Structured-illumination microscopy can double the resolution of the wide-field fluorescence microscope, but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed SIM that is capable of 100 nm resolution at frame rates up to 11 Hz for several hundred time frames. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila S2 cells in the total internal reflection (TIRF) mode.

The eukaryotic spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores and prevents anaphase onset until all kinetochores are aligned on the metaphase plate. In higher eukaryotes, cytoplasmic dynein is involved in silencing the SAC by removing the checkpoint proteins Mad2 and the Rod–Zw10–Zwilch complex (RZZ) from aligned kinetochores (Howell, B.J., B.F. McEwen, J.C. Canman, D.B. Hoffman, E.M. Farrar, C.L. Rieder, and E.D. Salmon. 2001. J. Cell Biol. 155:1159–1172; Wojcik, E., R. Basto, M. Serr, F. Scaerou, R. Karess, and T. Hays. 2001. Nat. Cell Biol. 3:1001–1007). Using a high throughput RNA interference screen in Drosophila melanogaster S2 cells, we have identified a new protein (Spindly) that accumulates on unattached kinetochores and is required for silencing the SAC. After the depletion of Spindly, dynein cannot target to kinetochores, and, as a result, cells arrest in metaphase with high levels of kinetochore-bound Mad2 and RZZ. We also identified a human homologue of Spindly that serves a similar function. However, dynein's nonkinetochore functions are unaffected by Spindly depletion. Our findings indicate that Spindly is a novel regulator of mitotic dynein, functioning specifically to target dynein to kinetochores.

Nucleoporin 98 (Nup98), a glycine-leucine-phenylalanine-glycine (GLFG) amino acid repeatcontaining nucleoporin, plays a critical part in nuclear trafficking. Injection of antibodies to Nup98 into the nucleus blocks the export of most RNAs. Nup98 contains binding sites for several transport factors; however, the mechanism by which this nucleoporin functions has remained unclear. Multiple subcellular localizations have been suggested for Nup98. Here we show that Nup98 is indeed found both at the nuclear pore complex and within the nucleus. Inside the nucleus, Nup98 associates with a novel nuclear structure that we term the GLFG body because the GLFG domain of Nup98 is required for targeting to this structure. Photobleaching of green fluorescent protein-Nup98 in living cells reveals that Nup98 is mobile and moves between these different localizations. The rate of recovery after photobleaching indicates that Nup98 interacts with other, less mobile, components in the nucleoplasm. Strikingly, given the previous link to nuclear export, the mobility of Nup98 within the nucleus and at the pore is dependent on ongoing transcription by RNA polymerases I and II. These data give rise to a model in which Nup98 aids in direction of RNAs to the nuclear pore and provide the first potential mechanism for the role of a mobile nucleoporin. INTRODUCTIONThe nuclear pore complex is a massive structure that conducts all traffic between the nucleus and cytoplasm (reviewed by Ohno et al., 1998;Gorlich and Kutay, 1999;Ryan and Wente, 2000;Vasu and Forbes, 2001). The pore has been studied intensely at the structural level in both yeast and vertebrate systems (Stoffler et al., 1999a;Allen et al., 2000). Although the vertebrate pore is larger and thought to contain a greater number of constituent proteins, the pores of both yeast and vertebrates share a similar structural organization. The central mass of the nuclear pore displays eightfold symmetry around a central axis perpendicular to the nuclear envelope. Two distinct sets of fibers extend out from the cytoplasmic and nuclear faces of the pore. On the nuclear side, the fibers are joined together at their distal ends by a ring to form the nuclear basket of the pore. Additionally, the nuclear basket of both yeast and vertebrate pores has associated filaments that extend for considerable distances into the nuclear interior and may serve to direct transport cargoes to and/or from the pore.A subset of the nuclear pore complex proteins (nucleoporins or Nups) each contain a domain with multiple, nontandem repeats of the amino acid sequences FG, FXFG, or GLFG (glycine-leucine-phenylalanine-glycine). These domains provide docking sites for a family of nuclear transport signal receptor proteins (known as importins, exportins, and transportin, or collectively as karyopherins). The repeat domain nucleoporins are found in multiple substructures of the nuclear pore and thus are thought to facilitate the moveArticle published online ahead of print. Mol. Biol. Cell 10.1091/ mbc.01-11-0538. Article and p...