Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Delta mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.

Proteins encapsulated within the aqueous core of reverse micelles are found to partially align in a magnetic field. The degree of alignment is sufficient to result in sizeable residual 15 N-1 H dipolar couplings that can be easily measured. It is found that the magnetic susceptibility of the reverse micelle particle is not dominated by the encapsulated protein. The residual dipolar couplings are found to be structurally meaningful.With the completion of several grand scale genome sequencing efforts, it has become possible to undertake a comprehensive analysis of the structural basis for the function of the proteins encoded by the human and other genomes. 1 The sheer number of proteins involved is daunting. 2 The task is made even more difficult by the observation that a significant fraction of the proteomes of various species is somewhat ill-suited for analysis by the two main structural methods: X-ray crystallography and solution NMR spectroscopy. 1-4 In particular, proteins of marginal stability in vitro are problematic for both approaches. In addition, solution NMR spectroscopy is somewhat limited by the relaxation properties of slowly tumbling macromolecules. One approach is to employ extensive deuteration and the TROSY-effect. 5 Another approach actively seeks to increase the effective rate of molecular reorientation by encapsulating the protein of interest within the protective shell of a reverse micelle and dissolving the resulting particle in a low viscosity fluid. 6 This method also allows the study of marginally stable proteins where the confined space of the reverse micelle is used to stabilize the compact native state. 7Human ubiquitin is the only example of a structure of an encapsulated protein determined to high resolution. 8 A current significant deficiency for structure determinations of encapsulated proteins has been the absence of longer range restraints derived from residual dipolar couplings (RDCs) arising from partial alignment of the protein in the magnetic field. 9,10 RDCs are an extremely powerful structural restraint. 11,12 Here we report that encapsulated proteins partially align within a magnetic field.We have examined the magnetically-induced alignment of three encapsulated proteins (Table 1). The apparent splittings (J+D) in the IPAP 15 N-1 H HSQC spectra were measured 13 at 17.6 Tesla and 11.7 Tesla. Encapsulated ubiquitin gave RDCs that were small (−0.9 to +0.5 Hz) but still ~5 fold greater than those measured in aqueous solution 14 . Encapsulated cytochrome c (cyt c), both the paramagnetic (oxidized) and diamagnetic (reduced) states, and oxidized (diamagnetic) encapsulated flavodoxin all showed significant RDCs (Figure 1). The RDCs for encapsulated oxidized cytochrome c are roughly five times larger than those seen in free wand@mail.med.upenn.edu. A simple strategy was used to evaluate whether the measured RDCs were structurally meaningful. Previously determined structures were used as a starting point in the evaluation (Table I). Distance restraints were generated for a...