Khaire Nandkumar, Müller Rolf, Blau-Wasser Rosemarie, Eichinger Ludwig, Schleicher Michael, Rief Matthias, Holak Tad A, Noegel Angelika A
Institut für Biochemie I, Zentrum Molekulare Medizin Köln, Medizinische Fakultät, Universität zu Köln, 50931 Köln, Germany.
J Biol Chem. 2007 Jan 19;282(3):1948-55. doi: 10.1074/jbc.M610262200. Epub 2006 Nov 22.
Dictyostelium strains lacking the F-actin cross-linking protein filamin (ddFLN) have a severe phototaxis defect at the multicellular slug stage. Filamins are rod-shaped homodimers that cross-link the actin cytoskeleton into highly viscous, orthogonal networks. Each monomer chain of filamin is comprised of an F-actin-binding domain and a rod domain. In rescue experiments only intact filamin re-established correct phototaxis in filamin minus mutants, whereas C-terminally truncated filamin proteins that had lost the dimerization domain and molecules lacking internal repeats but retaining the dimerization domain did not rescue the phototaxis defect. Deletion of individual rod repeats also changed their subcellular localization, and mutant filamins in general were less enriched at the cell cortex as compared with the full-length protein and were increasingly present in the cytoplasm. For correct phototaxis ddFLN is only required at the tip of the slug because expression under control of the cell type-specific extracellular-matrix protein A (ecmA) promoter and mixing experiments with wild type cells supported phototactic orientation. Likewise, in chimeric slugs wild type cells were primarily found at the tip of the slug, which acts as an organizer in Dictyostelium morphogenesis.
缺乏F-肌动蛋白交联蛋白细丝蛋白（ddFLN）的盘基网柄菌菌株在多细胞蛞蝓阶段存在严重的趋光性缺陷。细丝蛋白是杆状同型二聚体，可将肌动蛋白细胞骨架交联成高粘性的正交网络。细丝蛋白的每个单体链由一个F-肌动蛋白结合结构域和一个杆状结构域组成。在拯救实验中，只有完整的细丝蛋白能在细丝蛋白缺失突变体中重新建立正确的趋光性，而失去二聚化结构域的C端截短的细丝蛋白以及缺乏内部重复序列但保留二聚化结构域的分子均不能拯救趋光性缺陷。单个杆状重复序列的缺失也改变了它们的亚细胞定位，与全长蛋白相比，突变的细丝蛋白在细胞皮层的富集程度一般较低，且在细胞质中的含量越来越高。对于正确的趋光性，ddFLN仅在蛞蝓的顶端是必需的，因为在细胞类型特异性细胞外基质蛋白A（ecmA）启动子的控制下表达以及与野生型细胞的混合实验支持趋光定向。同样，在嵌合蛞蝓中，野生型细胞主要位于蛞蝓的顶端，蛞蝓顶端在盘基网柄菌形态发生中起组织者的作用。
