Structure And Function Of Unconventional Myosins

关于分子马达如何与它们的囊泡货物结合，我们知之甚少。我们现在已经表明，肌球蛋白Va，肌动蛋白为基础的囊泡电机，结合到它的货物之一，黑素体，通过与受体蛋白复合物含有Rab 27 a和黑素，一个假定Rab 27 a效应。Rab 27 a首先与黑素体结合，然后招募亲黑素蛋白，亲黑素蛋白又招募肌球蛋白Va。亲黑素通过其氨基末端以GTP依赖性方式与Rab 27 a结合，并通过其羧基末端与肌球蛋白Va结合来产生这种连接。后一种相互作用，类似于肌球蛋白Va与黑素体共定位并影响其在体内分布的能力，绝对依赖于外显子F的存在，外显子F是肌球蛋白Va尾中的选择性剪接外显子。这些结果提供了第一个肌动蛋白为基础的电机的细胞器电机的分子描述，说明如何交替使用外显子可以用来指定货物，并进一步扩大了功能库的Rab GTP酶及其效应。 肌动蛋白结合蛋白对肌动蛋白丝动力学的调节对于细胞的运动性、形状和结构至关重要。网骨藻蛋白CARMIL（用于加帽蛋白，ARp 2/3复合物，肌球蛋白I连接物）似乎用作连接三种不同肌动蛋白结合蛋白的支架：加帽蛋白（CP），肌动蛋白丝生长的主要终止子，Arp 2/3复合物，肌动蛋白丝生长的主要成核剂，和肌球蛋白I，一种普遍存在的有倒钩末端定向运动蛋白。在体内，CARMIL与CP、Arp 2/3和肌球蛋白I共定位于动态肌动蛋白丰富的延伸区，如迁移细胞和背侧巨胞饮结构的前缘。此外，在缺乏CARMIL的网骨藻细胞中，胞饮作用和趋化性聚集受到严重抑制，细胞内F-肌动蛋白含量降低。这些观察结果表明，CARMIL，这也是存在于脊椎动物细胞，是生理上重要的。为了表征CARMIL的生物化学特性，我们现在已经从阿米巴中纯化了它的同系物。关于纯化的最引人注目的观察是CP与CARMIL广泛共纯化。通过凝胶过滤在低pH下实现CARMIL和CP的分离。凝胶过滤研究还表明，CARMIL必须具有细长的形状或存在于多聚体中，因为洗脱体积反映了比CARMIL高得多的分子量？s序列意味着。与这些观察结果相一致，化学交联和分析超离心表明，纯化的CARMIL是在一个单体-二聚体平衡的缔合常数为10？六米？1，CP促进二聚化，并且CARMIL二聚体非常不对称。纯化的CP以每个CARMIL二聚体一个CP的化学计量重新结合CARMIL，并且亲和力为400 nM或更高。鉴于CARMIL和CP的细胞浓度（分别为2和1微摩尔），CP与CARMIL的结合可显著影响体内肌动蛋白丝的生长。重要的是，体外实验表明，CP-CARMIL复合物仍然可以覆盖肌动蛋白丝的倒刺末端。总之，这些结果表明CARMIL是真正的CP相互作用物，并且它们的相互作用不用于简单地螯合CP。鉴于CARMIL也与肌球蛋白I和Arp 2/3相互作用，我们认为CP-CARMIL复合物可能被肌球蛋白I易位到倒刺末端，并通过其招募Arp 2/3的能力，引发新的细丝从加帽的细丝末端聚合。

Little is known about how molecular motors bind to their vesicular cargo. We have now shown that myosin Va, an actin-based vesicle motor, binds to one of its cargoes, the melanosome, by interacting with a receptor-protein complex containing Rab27a and melanophilin, a postulated Rab27a effector. Rab27a binds to the melanosome first and then recruits melanophilin, which in turn recruits myosin Va. Melanophilin creates this link by binding to Rab27a in a GTP-dependent fashion through its amino terminus, and to myosin Va through its carboxy terminus. This latter interaction, similar to the ability of myosin Va to colocalize with melanosomes and influence their distribution in vivo, is absolutely dependent on the presence of exon-F, an alternatively spliced exon in the myosin Va tail. These results have provided the first molecular description of an organelle motor for an actin-based motor, illustrated how alternate exon usage can be used to specify cargo, and further expanded the functional repertiore of Rab GTPases and their effectors. Regulation of actin filament dynamics by actin binding proteins is critical for the motility, shape, and structure of cells. The Dictyostelium protein CARMIL (for Capping protein, ARp2/3 complex, Myosin I Linker) appears to serve as a scaffold that links three different actin binding proteins: capping protein (CP), the major terminator of actin filament growth, the Arp2/3 complex, the major nucleator of actin filament growth, and myosin I, a ubiquitous barbed-end-directed motor protein. In vivo CARMIL co-localizes with CP, Arp2/3, and myosin I in dynamic actin-rich extensions like the leading edge of migrating cells and dorsal macropinocytic structures. Furthermore, in Dictyostelium cells lacking CARMIL, pinocytosis and chemotactic aggregation are severely inhibited and the cellular content of F-actin is decreased. These observations suggest that CARMIL, which is also present in vertebrate cells, is physiologically important. To characterize CARMIL biochemically we have now purified its homolog from Acanthamoeba. The most striking observation regarding the purification is that CP copurifies extensively with CARMIL. Separation of CARMIL and CP was achieved by gel filtration at low pH. Gel filtration studies also suggested that CARMIL must either have an elongated shape or exist in multimers, since elution volumes reflected a much higher molecular weight than CARMIL?s sequence implies. Consistent with these observations, chemical crosslinking and analytical ultra centrifugation showed that purified CARMIL is in a monomer-dimer equilibrium with an association constant of 10?6M?1, that CP promotes dimerization, and that the CARMIL dimer is very asymmetric. Purified CP rebinds to CARMIL with a stochiometry of one CP per CARMIL dimer, and with an affinity of 400 nM or tighter. Given the cellular concentrations of CARMIL and CP (2 and 1 micromolar, respectively), binding of CP to CARMIL could significantly affect the growth of actin filaments in vivo. Importantly, in vitro experiments showed that the CP-CARMIL complex can still cap the barbed ends of actin filaments. Together, these results indicate that CARMIL is a bonafide CP interacter, and that their interaction does not serve to simply sequester CP. Given that CARMIL also interacts with myosin I and Arp2/3, we suggest that the CP-CARMIL complex may be translocated to the barbed end by myosin I, and through its ability to recruit Arp2/3, initiate polymerization of a new filament off of a capped filament end.