title: In vitro assay of neurofilament light chain self-assembly using truncated mutants.
abstract: Neurofilaments (NFs) are heteropolymers composed of light (NF-L), middle (NF-M), and heavy (NF-H) subunits, present in most neurons. NF-L polymerizes on its own to provide a scaffold on which regular NFs form via the cross-bridging of NF-M or NF-H. To clarify the mechanism of regulation of NF-L self-assembly, we developed an assay using truncated mutant NF-L fused to glutathione-S transferase (GST). Western immunoblotting data show that the GST-fused head-rod domains of NF-L are necessary and sufficient for detecting assembled NF-L. The levels of self-assembled NF-L subunits detected using GST fusion proteins were consistent with those detected by electron microscopy and turbidity assay. Our results collectively imply that GST-fused head-rod domains of NF-L are critical tools for analyzing NF-L self-assembly in vitro.
authors.affiliation: Department of Life Science, College of Natural Science, Daejin University, Kyeonggido 487-711, Republic of Korea.
authors.forename: Sung-Kuk
authors.initials: SK
authors.lastname: Kim
authors.forename: Sang-Min
authors.initials: SM
authors.lastname: Cho
authors.forename: In-Bum
authors.initials: IB
authors.lastname: Lee
authors.forename: Young Han
authors.initials: YH
authors.lastname: Lee
authors.forename: Jung Hoon
authors.initials: JH
authors.lastname: Kang
authors.forename: Jang Hyun
authors.initials: JH
authors.lastname: Choi
authors.forename: Pann-Ghill
authors.initials: PG
authors.lastname: Suh
authors.forename: Jong-Soo
authors.initials: JS
authors.lastname: Chang
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism* / Q000648:ultrastructure*; D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D045744:Cell Line, Tumor; D006801:Humans; D016297:Mutagenesis, Site-Directed; D016900:Neurofilament Proteins / Q000378:metabolism* / Q000648:ultrastructure*; D016716:PC12 Cells; D051381:Rats; D013329:Structure-Activity Relationship
chemical_list: D016900:Neurofilament Proteins; C044882:neurofilament protein L; C051774:neurofilament protein H