Structure And Function Of Unconventional Myosins

缺乏GTPase Rab27a（灰色）的黑素细胞在肌凝蛋白va依赖性黑素小体捕获中被禁用，因为肌凝蛋白与黑素小体表面的结合依赖于这种常驻黑素小体膜蛋白的存在。这些观察的一种解释是Rab27a作为重要组成部分的黑素体肌凝蛋白受体。我们已经表明,肌凝蛋白的能力Va尾巴域向黑素体本地化和生成肌凝蛋白Va null(稀释)在野生型表型melanocyes absoltutely依赖的存在外显子F两种或者拼接外显子存在于肌凝蛋白Va的melanocyte-spliced对碘氧基苯甲醚的尾巴而不是brain-spliced同种型。另一个黑色素细胞特异性尾外显子D则不需要。同样，全长肌凝蛋白Va与黑素小体共定位并挽救稀黑素细胞中黑素小体分布的能力完全依赖于外显子F的存在，因为黑素细胞肌凝蛋白Va和不含外显子D的黑素细胞肌凝蛋白Va可共定位并挽救，而不含外显子F的黑素细胞肌凝蛋白Va和脑肌凝蛋白Va不能共定位或挽救。这些结果表明，如果肌凝蛋白Va和Rab27a是一对运动受体，它们的物理关联应该依赖于外显子f。与此一致的是，存在于黑素细胞清洁剂裂解液中的Rab27a与纯化的、全长的、flag标记的黑素细胞肌球蛋白Va和缺乏外显子D的黑素细胞肌球蛋白Va结合，但不与缺乏外显子F的黑素细胞肌球蛋白Va结合，也不与脑肌球蛋白Va结合。此外，在GTP存在的情况下制备裂解液，使Rab27a与黑素细胞肌球蛋白Va结合的量减少了约4倍。最后，肌凝蛋白Va和Rab27a的稳定相互作用似乎需要至少一个额外的裂解物衍生因子，因为纯化的、gtp负载的Rab27a不能明显地与肌凝蛋白Va包被的珠结合。总之，这些结果坚定地证实了肌凝蛋白Va和Rab27a在黑素小体中作为马达：受体对发挥作用，表明它们的相互作用需要外显子F和至少一个额外的蛋白质，并表明肌凝蛋白Va在黑素小体表面的募集应该受到控制Rab27a核苷酸状态的因子的调节。Dictyostelium CARMIL蛋白作为支架，通过其SH3结构域将capping蛋白（CP）和Arp2/3复合物连接到I型肌球蛋白。为了进一步表征该复合物，我们纯化了棘阿米巴CARMIL以达到均匀性。分析性超离心、电子显微镜和化学交联研究表明，CARMIL处于单体：二聚体平衡状态，缔合常数为~ 1uM。关于CARMIL的纯化，最引人注目的观察是它与CP广泛地分离。通过凝胶过滤完全分离两者需要温和的混沌剂或低pH值（5.4）。纯化的CP与CARMIL再结合，每个CARMIL单体最大化学计量为2个CP异二聚体，亲和度为~ 100 nM。考虑到这种亲和性，以及CARMIL （~2 uM）和CP （~1 uM）的细胞浓度，CARMIL可以在体内显著影响倒刺端盖。最近的体外实验表明，CARMIL: CP复合物可以盖住肌动蛋白丝，这表明CARMIL的作用不是简单地将CP隔离在非活性池中。相反，CARMIL和CP形成了一个新的有倒刺的端帽，(i)可能被肌凝蛋白i转移到有倒刺的端上，（ii）可能通过其招募和激活Arp2/3的能力，允许新的肌动蛋白丝从有帽端组装起来。

Melanocytes that lack the GTPase Rab27a (ashen) are disabled in myosin Va-dependent melanosome capture because the association of the myosin with the melanosome surface is dependent on the presence of this resident melanosomal membrane protein. One interpretation of these observations is that Rab27a serves as an essential component of the melanosome receptor for myosin Va. We have now shown that the ability of the myosin Va tail domain to localize to the melanosome and generate a myosin Va null(dilute) phenotype in wild type melanocyes is absoltutely dependent on the presence of Exon F, one of two alternatively spliced exons present in the tail of the melanocyte-spliced isoform of myosin Va but not the brain-spliced isoform. Exon D, the other melanocyte-specific tail exon, is not required. Similarly, the ability of full-length myosin Va to colocalize with melanosomes and to rescue melanosome distribution in dilute melanocytes is absolutely dependent on the presence of Exon F, since melanocyte myosin Va, and melanocyte myosin Va without Exon D colocalize and rescue, while melanocyte myosin Va without Exon F and brain myosin Va do not colocalize or rescue. These results imply that if myosin Va and Rab27a are a motor: receptor pair, their physical association should be Exon F-dependent. Consistent with this, Rab27a present in melanocyte detergent lysates binds to beads coated with purified, full-length, FLAG-tagged melanocyte myosin Va and melanocyte myosin Va lacking Exon D, but not to melanocyte myosin Va lacking Exon F, or to brain myosin Va. Furthermore, the preparation of lysates in the presence of GDP rather than GTP reduces the amount of Rab27a bound to melanocyte myosin Va by ~4 fold. Finally, the stable interaction of myosin Va and Rab27a appears to require at least one additional lysate-derived factor, since purified, GTP-loaded Rab27a does not bind appreciably to myosin Va-coated beads. Together, these results firmly establish that myosin Va and Rab27a function in the context of the melanosome as a motor: receptor pair, show that their interaction requires Exon F and at least one additional protein, and suggest that the recruitment of myosin Va on to the melanosome surface should be regulated by factors controlling the nucleotide state of Rab27a. The Dictyostelium CARMIL protein acts as a scaffold to link capping protein (CP) and the Arp2/3 complex to type I myosins through their SH3 domains. To further characterize this complex, we have purified Acanthamoeba CARMIL to homogeneity. Analytical ultracentrifugation, electron microscopy, and chemical crosslinking studies show that CARMIL is in a monomer: dimer equilibrium with an association constant of ~ 1uM. The most striking observation regarding the purification of CARMIL is that it copurifies extensively with CP. Complete dissociation of the two by gel filtration requires a mild chaotropic agent or low pH (5.4). Purified CP rebinds to CARMIL with a maximum stoichiometry of two CP heterodimers per CARMIL momomer, and with an affinity of ~ 100 nM. Given this affinity, and the cellular concentrations of CARMIL (~2 uM) and CP (~1 uM), CARMIL would be predicted to significantly influence barbed end capping in vivo. Recent in vitro experiments show that the CARMIL: CP complex can cap actin filaments, indicating that CARMIL does not function to simply sequester CP in an inactive pool. Rather, CARMIL and CP form a novel barbed end cap that (i)may be translocated to barbed ends by myosin I, and (ii) may, through its ability to also recruit and activate Arp2/3, allow the assembly of a new actin filament off of a capped end.