Dysferlin Interacts with Tubulin and Microtubules in Mouse Skeletal Muscle
Dysferlin Interacts with Tubulin and Microtubules in Mouse Skeletal Muscle
复制标题
DOI：
10.1371/journal.pone.0010122
复制
发表时间：
2010-04-12
期刊：
影响因子：
3.7
通讯作者：
Sinnreich, Michael
中科院分区：
文献类型：
--
作者：
Azakir, Bilal A.;Di Fulvio, Sabrina;Sinnreich, Michael
迪弗林是一种II型跨膜蛋白，与肌肉的表膜修复有关。Deferlin基因突变导致肢体带状肌营养不良2B、Miyoshi肌病和远端前筋膜室肌病。迪弗林的作用模式还不是很清楚，到目前为止，只有几个蛋白质结合伙伴被确定。通过亲和纯化和液-质联用，我们确定了α-微管蛋白是一种新的去铁蛋白的结合伙伴。体外谷胱甘肽S转移酶下拉实验和微管结合实验证实了去铁蛋白与α-微管蛋白以及去铁蛋白与微管的相互作用。免疫共沉淀法在体内证实了这些相互作用。共聚焦显微镜显示，异铁蛋白和α-微管蛋白共同定位于成肌细胞的核周区和泡状结构中，并沿肌管中类似微管的细长结构分布。我们将dyferlin的α-微管蛋白结合区映射到它的C2a和C2b结构域。钙水平的调节不影响去铁蛋白与α-微管蛋白的结合，这表明这种相互作用是不依赖于钙的。我们的研究确定了一个新的结合性伙伴，并提示微管在将脱铁蛋白运输到肌膜中发挥作用。
Dysferlin is a type II transmembrane protein implicated in surface membrane repair in muscle. Mutations in dysferlin lead to limb girdle muscular dystrophy 2B, Miyoshi Myopathy and distal anterior compartment myopathy. Dysferlin's mode of action is not well understood and only a few protein binding partners have thus far been identified. Using affinity purification followed by liquid chromatography/mass spectrometry, we identified alpha-tubulin as a novel binding partner for dysferlin. The association between dysferlin and alpha-tubulin, as well as between dysferlin and microtubules, was confirmed in vitro by glutathione S-transferase pulldown and microtubule binding assays. These interactions were confirmed in vivo by co-immunoprecipitation. Confocal microscopy revealed that dysferlin and alpha-tubulin co-localized in the perinuclear region and in vesicular structures in myoblasts, and along thin longitudinal structures reminiscent of microtubules in myotubes. We mapped dysferlin's alpha-tubulin-binding region to its C2A and C2B domains. Modulation of calcium levels did not affect dysferlin binding to alpha-tubulin, suggesting that this interaction is calcium-independent. Our studies identified a new binding partner for dysferlin and suggest a role for microtubules in dysferlin trafficking to the sarcolemma.