CRISPR/Cas9-mediated deletion of the Wiskott-Aldrich syndrome locus causes actin cytoskeleton disorganization in murine erythroleukemia cells

PeerJ

article

Vanessa Fernández-Calleja¹  María-José Fernández-Nestosa²  Pablo Hernández¹  Jorge B. Schvartzman¹  Dora B. Krimer¹ 

[1] Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, Spanish National Research Council;Bioinformatic Laboratory, Polytechnic School, National University of Asuncion

关键词: Wiskott-Aldrich;    Erythroleukemia cells;    Actin cytoskeleton;    CRISPR/Cas9;    Bruton tyrosine kinase;   

DOI  :  10.7717/peerj.6284

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

Wiskott-Aldrich syndrome (WAS) is a recessive X-linked inmmunodeficiency caused by loss-of-function mutations in the gene encoding the WAS protein (WASp). WASp plays an important role in the polymerization of the actin cytoskeleton in hematopoietic cells through activation of the Arp2/3 complex. In a previous study, we found that actin cytoskeleton proteins, including WASp, were silenced in murine erythroleukemia cells defective in differentiation. Here, we designed a CRISPR/Cas9 strategy to delete a 9.5-kb genomic region encompassing the Was gene in the X chromosome of murine erythroleukemia (MEL) cells. We show that Was-deficient MEL cells have a poor organization of the actin cytoskeleton that can be recovered by restoring Was expression. We found that whereas the total amount of actin protein was similar between wild-type and Was knockout MEL cells, the latter exhibited an altered ratio of monomeric G-actin to polymeric F-actin. We also demonstrate that Was overexpression can mediate the activation of Bruton’s tyrosine kinase. Overall, these findings support the role of WASp as a key regulator of F-actin in erythroid cells.

【 授权许可】

CC BY