Two-dimensional distribution and dynamics of membrane proteins and lipids revealed by freeze-fracture replica immuno-electron microscopy

s35

ROLE AND MODE OF ACTION OF DOC2 Ih’ NEUROTRANSMITTER OF POSTSYNAPTIC LONG-TERM POTENTIATION

MS04-3 YOSHIMI

TAKAI’.

TOSHIYA

M.4NABE’.

TAKUYA

SASAKI’.

KATSUNORI

SATOSHI

ORITA’.

KOBAYASHI’.

‘Dept. of Mol. Biol. and Biochem.. Osaha Univ. Medical ?;66-0022. ‘Dept. ofNeurophqs.. Fat. of Medicine. Unik.

RELEASE

GAKU SAKAGUCHI’.

SUMIKO

is specifically

expressed

whereas Doc2P is ubiquitously a diacylglycerol-

increases

manner. and this DocZa-Munc13

interactions

the efficacy for the recruitment

plasma membrane.

cause recruitment

a

in

of synaptic

study using the hippocampal

mice indicate that Doc2a is not essential for the Ca’+-dependent

increased efficacy causes continuous

of two isoforms,

on synaptic vesicles at nerve terminals,

vesicles from the reserve pool to the readily releasable pool. The electrophysiological region of DocZa-deficient

Doc2 consists

Doc2a interacts with Munc 13. localized on the presynaptic

or phorbol ester-dependent

TAKAHASH13,

:Shionogi Institute for Medical Science, Settsu. Osaka of Phys.. Tokyo Medical College, Tokyo t60-8102

School, Suita. Osaka 56.5-0871. of Tokyo. Tokyo I l3-0033,‘Dept.

in neural tissue where it is concentrated

expressed.

TOMOYUKI

MOCHlDA”

Doc2 is a protein having two C2 domains interacting with Ca)+ and phospholipid. and p, Doc2a

AND INDUCTION

CA1

glutamate release process hut

of synaptic vesicles from the reserve pool to the readily releasable

and sufficient release of glutamate which then induces postsynaptic

pool and that this

long-term

potentiation. TWO-DIMENSIONAL

MS04-4 KAZUSHI

REVEALED

BY FREEZE-FRACTURE

Recently, we developed freeze-fracture

exoplasmic

SDS-FRL

OF MEMBRANE

IMMUNO-ELECTRON

PROTEINS

AND LIPIDS

MICROSCOPY

Kyoto Univ., Kyoto 606-8501

replica labeling (SDS-FRL)

unfixed, quick-frozen dissolves

unfractured

membrane

to study the two-dimensional

of this distribution

and platinum/carbon

areas of the cell membranes,

replication,

sodium dodecyl sulfate (SDS)-digested

distribution

to images of freeze-fracture

cells, after freeze-fracture

with the specific antibody.

of the cytochemical

labeling on the mem-

replicas created by platinum shadowing. (Pt/C) shadowing,

with the release of the cytoplasmic

which are indistinguishable

Integral membrane

membranes.

contents.

The cytoplasmic

to labeling of various membrane

proteins and phospholipds.

and

proteins revealed as intramembrane

on a pure morphological

In this presentation,

In

are treated with SDS. The

basis, can be selectively

In addition, this approach can be applied to examine the transmembrane

in various cell and intracellular

its application

replica labeling technique,

surfaces can be then labeled cytochemically.

particles by freeze-fracture

distribution

REPLICA

a quick-freezing/freeze-fracture

brane surface and the relationship

detergent

AND DYNAMICS

FUJIMOTO

Dept. of Anatomy, Fat. of Medicine,

SDS-FRL.

DISTRIBUTION

labeled by

phospholipid

I introduce the practical procedure for SDS-FRL,