Dephosphorylation of the largest neurofilament subunit protein influences the structure of crossbridges in reassembled neurofilaments.
Phosphorylation-dependent change in electrophoretic mobility is the most unique characteristic of NF-H, the largest molecular mass subunit of the neurofilament. We dephosphorylated NF-H using Escherichia coli alkaline phosphatase, then reassembled it into neurofilaments with NF-M and NF-L, and into NF-H filaments with NF-H alone. We compared these dephosphorylated filaments with control: projections by low-angle rotary-shadow, crossbridges by quick-freeze deep-etch, and core filament packing density by thin-section electron microscopy. Projections in the dephosphorylated filaments were basically similar in structure to those in control, although there was a tendency for them to be wider and less dense, especially in NF-H filaments. Dephosphorylated filaments were still able to form crossbridges between core filaments, but their crossbridges were significantly wider, less dense, more branched and more irregular than crossbridges in control, and core filaments were more densely packed. These structural differences may be brought about by the removal of phosphate groups from NF-H tail and consequent reduction of electrostatic repulsion between adjacent crossbridges extending from the same core filament. The results indicate that phosphorylation of NF-H is necessary for forming well developed crossbridges, straight and at constant intervals, like those of in vivo axonal neurofilaments.
Gotow
T
T
Department of Anatomy, Osaka University Medical School, Japan.
Tanaka
T
T
Nakamura
Y
Y
Takeda
M
M
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005613:Freeze Etching; D008854:Microscopy, Electron; D016900:Neurofilament Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure*; D010766:Phosphorylation; D013116:Spinal Cord / Q000378:metabolism
D016900:Neurofilament Proteins; C051774:neurofilament protein H