F-actin clustering and cell dysmotility induced by the pathological W148R missense mu-

1 tation of filamin B at the actin-binding domain. 2 3 Yongtong Zhao, Sandor S. Shapiro† and Masumi Eto 4 5 Department of Molecular Physiology and Biophysics, Sidney Kimmel Medical College at Thomas Jef6 ferson University, and Sidney Kimmel Cancer Center, 1020 Locust Street, 436 Jefferson Alumni Hall, 7 Philadelphia, Pennsylvania 19107, USA 8 9 Running Head: Human filamin B mutations in cell dysmotility 10 11 *Corresponding to: Masumi Eto Email: [email protected] 12 13 14 Filamin B (FLNB) is a dimeric actin-binding pro15 tein that orchestrates the reorganization of the 16 actin cytoskeleton. Congenital mutations of 17 FLNB at the actin-binding domain (ABD) are 18 known to cause abnormalities of skeletal devel19 opment, such as atelosteogenesis type-I/III and 20 Larsen’s syndrome, although the underlying 21 mechanisms are poorly understood. Here, us22 ing fluorescence microscopy, we characterized 23 the reorganization of the actin cytoskeleton in 24 cells expressing each of 6 pathological FLNB 25 mutants that have been linked to skeletal ab26 normalities. The subfractionation assay showed 27 a greater accumulation of the FLNB ABD mu28 tants, W148R and E227K, to the cytoskeleton, 29 compared with the wild type protein. Ectopic 30 expression of FLNB-W148R, and the E227K 31 protein with a lesser extent, induced formation 32 of prominent F-actin accumulations, the conse33 quent rearrangements of focal adhesions, myo34 sin-II and septin filaments, and results in a de35 layed directional migration of the cells. The 36 W148R protein-induced cytoskeletal re37 arrangement was partially attenuated by the 38 inhibition of myosin-II, PAK or ROCK. The ex39 pression of a single-head ABD fragment with 40 the mutations partially mimicked the rear41 rangement induced by the dimer. The F-actin 42 clustering through the interaction with the mu43 tant FLNB ABD may limit the cytoskeletal reor44 ganization preventing normal skeletal develop45 ment. 46 47