Huber L A, Fialka I, Paiha K, Hunziker W, Sacks D B, Bähler M, Way M, Gagescu R, Gruenberg J
Research Institute of Molecular Pathology, I.M.P., Dr. Bohrgasse 7, A-1030 Wien, Austria.
Traffic. 2000 Jun;1(6):494-503. doi: 10.1034/j.1600-0854.2000.010607.x.
In epithelial cells, endocytosed transferrin and its receptor, which cycle basolaterally, have been shown to transit through recycling endosomes which can also be accessed by markers internalized from the apical surface. In this work, we have used an in vitro assay to follow transfer of an endocytosed marker from apical or basolateral early endosomes to recycling endosomes labeled with transferrin. We show that calmodulin (CaM) function is necessary for transfer and identified myr4, a member of the unconventional myosin superfamily known to use CaM as a light chain, as a possible target protein for CaM. Since myr4 is believed to act as an actin-based mechanoenzyme, we tested the role of polymerized actin in the assay. Our data show that conditions which either prevent actin polymerization or induce the breakdown of existing filaments strongly inhibit interactions between recycling endosomes and either set of early endosomes. Altogether, our data indicate that trafficking at early steps of the endocytic pathway in Madin-Darby Canine Kidney cells depends on the actin-based mechanoenzyme myr4, its light chain CaM, and polymerized actin.
在上皮细胞中，内化的转铁蛋白及其受体在基底外侧循环，已被证明可通过回收内体转运，而从顶端表面内化的标记物也可进入回收内体。在这项研究中，我们使用了一种体外测定法，来追踪内化标记物从顶端或基底外侧早期内体向用转铁蛋白标记的回收内体的转移。我们发现钙调蛋白（CaM）的功能对于这种转移是必需的，并确定了myr4，它是非常规肌球蛋白超家族的成员，已知以CaM作为轻链，可能是CaM的靶蛋白。由于myr4被认为是一种基于肌动蛋白的机械酶，我们在测定中测试了聚合肌动蛋白的作用。我们的数据表明，阻止肌动蛋白聚合或诱导现有细丝分解的条件会强烈抑制回收内体与任何一组早期内体之间的相互作用。总之，我们的数据表明，在Madin-Darby犬肾细胞内吞途径的早期步骤中的运输依赖于基于肌动蛋白的机械酶myr4、其轻链CaM和聚合肌动蛋白。
