The neuronal cytoskeleton-morphogenesis, organelle transport, and synaptic transmission

S5

II. Molecular architecture and function of the neuronal cytoskeleton

2. Symposia

THE N E U R O N A L C Y T O S K E L E T O N - M O R P H O G E N E S I S , O R G A N E L L E TRANSPORT, AND SYNAPTIC TRANSMISSION NOBUTAKA H I R O K A W A D e p a r t m e n t of A n a t o m y and Cell Biology, Medical School U n i v e r s i t y of Tokyo. Hongo, Tokyo, 113, Japan Neurons are highly p o l a r i z e d cells; that is they receive stimuli mainly at synaptic sites on dendrites and soma, integrate the stimuli at the dendrites and soma, and initiate and conduct these impulses through the axons. They develop c h a r a c t e r i s t i c 3 - d i m e n s i o n a l structures for this purpose. In this regard, the d i f f e r e n t i a t i o n of the cytoskeletal o r g a n i z a t i o n can be considered as providing a n e c e s s a r y base for the various functions w i t h i n nerve cells. We have been studying m o l e c u l a r architecture, dynamics, and function of neuronal c y t o s k e l e t o n s in special r e l a t i o n s h i p with morphogenesis, axonal transport, and synaptic transmission. A m o n g cytoskeletal proteins, we are interested in m i c r o t u b u l e a s s o c i a t e d proteins (MAPs, MAPIA, MAPIB, MAP2, Tau) n e u r o f i l a m e n t triplet proteins, kinesin, brain dynein, and synapsin I. We here present the m o l e c u l a r structure and i n t r a c e l l u l a r localization of these proteins in the neuronal c y t o s k e l e t o n using biochemistry, i m m u n o c y t o c h e m i s t r y , and quickfreeze deep-etch electron microscopy. We have studied the m e c h a n i s m of c o m p a r t m e n t a t i o n of MAP2 and tau (MAP2 in dendrites, tau in the axons) in cultured nerve cells by m i c r o i n j e c t i o n of labeled proteins and have found that the l o c a l i z a t i o n of these proteins are c o n t r o l l e d by local selective s t a b i l i z a t i o n mechanisms. Finally, we present the analysis of function of tau proteins in vivo by t r a n s f e c t i n g tau cDNA into fibroblasts which n o r m a l l y do not synthesize tau. We prove that tau p r o m o t e s tubulin p o ] y m e r i z a t i o n and forms m i c r o t u b u l e bundles in vivo, thus suggesting i m p o r t a n t roles in g r o w i n g and m a i n t a i n i n g nerve cell processes in vivo.

INVOLVEMENT OF CYTOSKELETAL ELEMENTSAND s r c GENE PRODUCTIN SYNAPTOGENESIS Kenji Sobue, Department o~f Neurochemistry an__~dNeuropharmacology, Biomedical Research Center, Osaka University Medical School, Nakanoshima, Kita-ku, Osaka 530, Japan We have used biochemical and immunocytochemical techniques to investigate the possible involvement of membrane cytoskeletal elements such as ~-actinin, calspectin (brain spectrin or fodrin), actin and

src

gene product (pp60c-src) in growth cone a c t i v i t i e s . During NGF-

induced d i f f e r e n t i a t i o n of PC12 cells,

~-actinin

increased

in

association with

neurite

outgrowth and was predominantly distributed throughout the entire growth cone and the distal portion of neurites.

Filopodial movements were sensitive to Ca2+ flux.

Two types of ~-

actinin, with Ca2+-sensitive and -insensitive actin binding a b i l i t i e s , were i d e n t i f i e d in the d i f f e r e n t i a t e d cells.

Ca2+-sensitive a-actinin

and actin

filaments were concentrated

in

filopodia. The Ca2+-insensitive protein was distributed from the body part of the growth cone to the distal portion of neurites, corresponding to substratum-adhesive sites. The expression of pp60c-src was also enhanced in association with

neurite outgrowth.

The locations of

calspectin and pp60c-src in growth cones were similar to those of the Ca2+-insensitive aactinin. These results are consistent with the hypothesis that Ca2+-sensitive ~-actinin and actin filaments are involved in Ca2+-dependent f i l o p o d i a l movement and Ca2+-insensitive eactinin, calspectin, and

pp60c - s r c

in adhesion of growth cones.