ngf-dependent protein kinase that phosphorylates maps . evidence for close relation of its activity...
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28. Neurocytology MORPHOMETRY OF THE DENDRITIC SPINES OF GRANULE CELLS IN RAT DENTATE GYRUS. Kiyoshi Hama, School of Human Sciences, Waseda University, Tokorozawa, 359, Japan:
Number, length, diameter and surface area of dendritic spines of granule cells in rat dentate gyrus were measured by three dimensional image analyzer using high voltage electron microscope stereo images of 5~m thick Golgi preparations. To increase accuracy of the analysis, we used an image intensifier with a real time image processor which permitted low electron beam observation, and a specially designed side entry specimen tilt stage with a feedback control mechanism for tilt angle. Number of spines was 1.5 to 2.0 times the previously reported light microscopical results. Although number of spines per unit length of dendrite was larger at the proximal portion of the dendrite and became smaller towards the distal portion, number of spines per unit surface area of the dendrite was about equal, 0.8 to 0.9 /~m 2, throughout the dendritic trees. The total dendritic surface area was doubled by addition of spine surfaces. The length of spines measured in three dimensions was about 1.4 times longer than that measured in two dimensions. More than 70 % of spines had a stalk less than 0.2 pm in diameter. We conclude that the resolution of the light microscope was insufficient for quantitative analysis of dendritic spines.
TAU PROTEINS: MOLECULAR STRUCTURE AND MODE OF BINDING ON MICROTUBULES. NOBUTAKA HIROKAWA r YOSHIMITSU KANAI r SHIGEO OKABE r Department of Anatom Z and Cell Biology, Medical School r University of Tokyo~ 7-3-1Hongo r Tokyo r 113 r Japan.--
Tau is a family of closely related proteins (55,000-62,000 mol w£) contained in nerve cells. They copolymerize with tubulin to induce formation of microtubules in vitro. All information so far has indicated that tau is closely apposed to the -- micr0tubule lattice, and there has been no indication of domains projecting from the microtubule polymer lattice. We studied the molecular structure of the tau factor and its mode of binding on microtubules using the quick-freeze, deep-etch method (QF DE) and low angle rotary shadowing. Phosphocellulose column-purified tubulin from porcine brain was polymerized with tau and the centrifuged pellets were processed by QF-DE. We observed periodic armlike elements (18.7 + 4.8 nm long) projecting from the microtubule surface. Most of the projections appeared to cross-link adjacent microtubules. We measured the longitudinal periodicity of tau projections on the microtubules and found it to match the 6-dimer pattern better than the 12-dimer pattern. The stoichiometry of tau versus tubulin in preparations of tau saturated microtubules was 1:-5.0 (molar ratio). Tau molecules adsorbed on mica took on rodlike forms (56.1 + 14.1 nm long). Although both tau and MAP1 are contained in axons, competitive binding studies demonstrated that the binding sites of tau and MAPIA on the microtubule surfaces are mostly distinct, although they may partially overlap.
NGF-DEPENDENT PROTEIN KINASE THAT PHOSPHORYLATES NAPS IN VITRO. EVIDE~'CE FOR CK)SE RELATION OF ITS ACTIVITY AND OUTGI¢OWTlt OF NEURITIS IN ~',,2D CELIa. MAMORU SANO, AND SATOK!) KlTAJIMA~l)e~artment of_ Y, orp,holoKv, lo.~t~!~gte f or_Devetopaen~3! P, [email protected], Alclji Colony, ga~ Kai, Aie)i_~!80-03~ Japan,
iJe have e s t ab l i shed a subline, oI ~ '12 c e i l s ([Ei21)) which exte.qd neurJ tes very quickly in response nut only to NGF but a l so to rANP !ike pr i ,~d c e l l s . [,, phosphorylat ion of brain MAP2 in the c e l l u l a r
exLracts , tuo d i s t i n c t kinase a c t i v i t i e s were ac t i va t ed (5-10 fold) orl b r i e f exposure of c e l l s to NGF or dbcAMP. The apparent molecular weiKht ol the kinase ac t iva ted by dbeAMP was 40Kda., which seems to be
A-kinase, and another a c t i v a t ~ i by NGF was 50Kda. The iat.Ler ~as m~xir, a l l y ac t iva ted in 5 rain on exposure to NGF, and was de tec tab le in the presence of Mg ~+, but did not rc~tuire Ms 2~ or Ca 2+ I t appears to be d i s t i n c t from previous ly reported k inascs in PC12 ce] Is. A pro te in kinase i n h i b i t o r , t(252a, s e l e e t i v e l y inh ib i t ed outgrowth of neu r i t e s in response to NGF but not to d~AMP. When t h i s inh ib i to r was added to the incubat ion of the c e l l s with NGF or dbcAMP, aet . ivation of the NGF-dependent kinase a c t i v i t y was s e l e c t i v e l y decreased. We isolatP-xt a few m~t~nt c lones from FE12D c e l l s which were d e f i c i e n t in the a b i l i t y to induce neu r i t e s in response to e i t h e r s t imula to r . In those va r i an t c e l l s , the a c t i v i t y of a p ro te in kinase corresponding to Lheir deficierd: neu r i t e response to NGF or dbcAMP was r~ lec t ive ly decreased. The NGF-dependent k inase might have an important ro le in outgrowth of n e u r i t e s from FE12 c e l l s in respons~e to NGF.