In vitro assay of neurofilament light chain self-assembly using truncated mutants.
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.
Kim
Sung-Kuk
SK
Department of Life Science, College of Natural Science, Daejin University, Kyeonggido 487-711, Republic of Korea.
Cho
Sang-Min
SM
Lee
In-Bum
IB
Lee
Young Han
YH
Kang
Jung Hoon
JH
Choi
Jang Hyun
JH
Suh
Pann-Ghill
PG
Chang
Jong-Soo
JS
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
D016900:Neurofilament Proteins; C044882:neurofilament protein L; C051774:neurofilament protein H