Journal of Neuroscience, Vol 9, 959-966, Copyright © 1989 by Society for Neuroscience
The effects of dephosphorylation on the structure of the projections of neurofilament
S Hisanaga and N Hirokawa
Department of Anatomy and Cell Biology, School of Medicine, University of Tokyo, Japan.
Carboxy-terminal tail domains of larger molecular mass subunits (NF-M and
NF-H) of neurofilaments (NFs), which are the highly phosphorylated
moieties, were observed as thin flexible filaments projecting from NF core
filaments by rotary shadowing (Hisanaga and Hirokawa, 1988).
Dephosphorylation of NFs has been suspected to affect the structures and
the functions of the carboxy-terminal tail projections. We report here the
effects of the dephosphorylation on the structure of NFs studied by
electron microscopy. (1) The structures of carboxy-terminal tail
projections after dephosphorylation were compared with those of the control
NFs by low-angle rotary shadowing. This was examined with 2 samples; the
isolated neurofilaments and the short filaments assembled from NF-H. Both
the dephosphorylated NFs and the short filaments showed many projections
laterally extending from core filaments similar to those observed in the
control samples. (2) With respect to the structure of NF in physiological
solution, the density of NFs in the precipitates was examined by
thin-section electron microscopy. No difference in the density was noted
between control and dephosphorylated NFs. (3) The ability to form
cross-bridges in vitro was examined by quick-freeze, deep-etch electron
microscopy. The structure and frequency of cross-bridges appeared to be
similar in both control and dephosphorylated NFs. (4) Phosphate
determination revealed that about 90% of the phosphate groups of NF-H
subunit were removed by treatment with E. coli alkaline phosphatase. These
results indicated that the dephosphorylation of NF did not affect the
structure and the ability to form cross-bridges of the carboxy-terminal
tail projections in vitro.
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