F-actin clustering and cell dysmotility induced by the pathological W148R missense mutation of filamin B at the actin-binding domain.
Filamin B (FLNB) is a dimeric actin-binding protein that orchestrates the reorganization of the actin cytoskeleton. Congenital mutations of FLNB at the actin-binding domain (ABD) are known to cause abnormalities of skeletal development, such as atelosteogenesis types I and III and Larsen's syndrome, although the underlying mechanisms are poorly understood. Here, using fluorescence microscopy, we characterized the reorganization of the actin cytoskeleton in cells expressing each of six pathological FLNB mutants that have been linked to skeletal abnormalities. The subfractionation assay showed a greater accumulation of the FLNB ABD mutants W148R and E227K than the wild-type protein to the cytoskeleton. Ectopic expression of FLNB-W148R and, to a lesser extent, FLNB-E227K induced prominent F-actin accumulations and the consequent rearrangement of focal adhesions, myosin II, and septin filaments and results in a delayed directional migration of the cells. The W148R protein-induced cytoskeletal rearrangement was partially attenuated by the inhibition of myosin II, p21-activated protein kinase, or Rho-associated protein kinase. The expression of a single-head ABD fragment with the mutations partially mimicked the rearrangement induced by the dimer. The F-actin clustering through the interaction with the mutant FLNB ABD may limit the cytoskeletal reorganization, preventing normal skeletal development.
Department of Molecular Physiology and Biophysics, Sidney Kimmel Medical College at Thomas Jefferson University, and Sidney Kimmel Cancer Center, Philadelphia, Pennsylvania.
Yongtong
Y
Zhao
Department of Molecular Physiology and Biophysics, Sidney Kimmel Medical College at Thomas Jefferson University, and Sidney Kimmel Cancer Center, Philadelphia, Pennsylvania.
Sandor S
SS
Shapiro
Department of Molecular Physiology and Biophysics, Sidney Kimmel Medical College at Thomas Jefferson University, and Sidney Kimmel Cancer Center, Philadelphia, Pennsylvania masumi.eto@jefferson.edu.
Masumi
M
Eto
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002465:Cell Movement / Q000187:drug effects; D064448:Filamins / Q000235:genetics* / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism; D005838:Genotype; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D024462:Myosin Type II / Q000037:antagonists & inhibitors / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D013997:Time Factors; D014162:Transfection; D054462:p21-Activated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D000199:Actins; C578662:FLNB protein, human; D064448:Filamins; D047428:Protein Kinase Inhibitors; D054462:p21-Activated Kinases; D054460:rho-Associated Kinases; D024462:Myosin Type II
F-actin; cell motility; cytoskeleton; fluorescence microscopy; skeletal dysplasia