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Journal of Neuroscience, Vol 8, 2769-2779, Copyright © 1988 by Society for Neuroscience
MAP2 is a component of crossbridges between microtubules and neurofilaments in the neuronal cytoskeleton: quick-freeze, deep-etch immunoelectron microscopy and reconstitution studies
N Hirokawa, S Hisanaga and Y Shiomura
Department of Anatomy and Cell Biology, School of Medicine, University of Tokyo, Japan.
Microtubules (MT) and neurofilaments (NF) are linked by frequent
crossbridges in situ. In order to answer the question of what makes these
crossbridges, we performed the immunogold procedure on rat spinal cord
motor neurons using an affinity-purified polyclonal antibody against rat
brain MAP2 and gold-labeled anti-rabbit IgG goat IgG. A quick-freeze,
deep-etch technique (QF-DE) in conjunction with decoration with anti-MAP2
antibody and ferritin-labeled second antibody was also used. In motor
neuron dendrites crossbridges were clearly displayed between MTs and NFs by
QF-DE. These crossbridges were revealed in thin sections as fuzzy
filamentous structures between MT and NF. Gold particles studded the fuzzy
structures associated with MT. Many such structures connected MTs to NFs.
Furthermore, antibody complexes containing ferritin were localized on the
crossbridges between MTs and NFs by the QF-DE study. In addition, we
performed reconstitution experiments. We isolated 70 kDa (L) protein of
neurofilaments from calf spinal cords and assembled L to form
neurofilaments in vitro. MAP2 bound these neurofilaments according to both
SDS-PAGE and QF-DE electron microscopy of the pellets of suspensions
containing L proteins and MAP2. When we added tubulin to this suspension
and polymerized it in the presence of taxol, neurofilaments were
crosslinked with microtubules by MAP2 crossbridges. Hence, from these 2
approaches we concluded that MAP2 is a component of crossbridges between
MTs and NFs in the neuronal cytoskeleton in vivo and in vitro.
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