The bundling of MTs by ase1p homologs requires the formation of protein oligomers to bring together two or more MT binding domains; homologs in budding yeast (Schuyler et al., 2003
) and plants (Smertenko et al., 2004) were proposed to form dimers, and the mammalian homolog PRC1 is believed to form even higher-order oligomers (Mollinari et al., 2002, Zhu et al., 2006). Dimer formation by the N terminus of ase1p (Figure 3C) and immunoprecipitation of HA-tagged ase1p and GFP-tagged ase1p (Figure 3D) showed that fission yeast ase1p oligomerizes. Preferential antiparallel bundling may therefore be enforced by a rigid antiparallel positioning of MT binding domains within ase1p oligomers. Interestingly, the N terminus of ase1p has a predicted spectrin domain (InterPro database) that was required for oligomerization (Figures S3 and Figure 3D). Spectrin repeats form antiparallel dimers in muscle α-actinin, which bundles antiparallel actin filaments in muscle Z disks (Djinovic-Carugo et al., 1999). The structural basis of MT and actin bundling may therefore be in part conserved.
Ase1p Autonomously Localizes to Interdigitated MTs
How do motor and bundling proteins like klp2p and ase1p target specific locations within MT networks? Similarly to kinesin-14 motors in other systems (Goshima et al., 2005, Sproul et al., 2005), klp2p may be recruited to MT plus ends by specialized plus-end-tracking proteins like EB1 or Cik1. Bundling proteins may depend on motor proteins for their localization; NuMA and PRC1 were proposed to associate with oppositely directed motors (dynein [Merdes et al., 1996] and Kif4 [Zhu et al., 2005]) for transport along MTs toward the spindle poles and the spindle midzone, respectively. Could a minus-end-directed motor transport ase1p along MTs toward antiparallel MTs in the bundle-midzone? The only minus-end-directed motor shown to affect bundle organization is, however, klp2p (Carazo-Salas et al., 2005), and ase1-GFP remains associated with antiparallel MTs in klp2Δ cells (Figure 2D and Movie S7). To investigate whether ase1p localizes autonomously to antiparallel MTs, we bundled polarity-marked stabilized MTs with bacterially expressed His-ase1-GFP. MTs were bound to the coverslip of a flowcell, and bundles with various amounts of MTs could be observed (Figure 3E). The majority of MTs in bundles of two MTs were antiparallel (66% ± 6%, ±SD, ntotal = 61), but the orientational preference was smaller than observed previously (Figure 3B). In the current assay, weak nonspecific interactions between ase1p and MTs may have bundled parallel MTs in solution because no large forces were required to bend MTs. The average fluorescence intensity of His-ase1-GFP along parallel MTs was 35% ± 8% (±SEM; n = 16) of that along antiparallel MTs (n = 28) (
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