電子顕微鏡による細胞内微細空間構造の解析：膜細胞骨格から核膜骨格へ

Spatial structure of membrane and perinuclear cytoskeletons revealed by electron
microscopyMy research works
Early part

University of Tokyo, School of Medicine
UCLA

Nagoya University, School of Medicine

Structures related to the photo-transduction fromthe outer segments to synapses

Later part

Nagoya University, School of Medicine
CRAST EcoTopia SI

Spatial structure of actin cytoskeletons frommembrane undercoat to peri-nucleus

Bird’s-eye view of frog retina by SEM

PEROSIS視細胞外節の超微構造
Freeze-substituted
thin section

30 nm

Fine structure of rod outer segmentsFreeze fracture replica

Supra molecular organization of rod
outer segments

PDETb

Freeze-etched replicas of rod outer
segments of photoreceptor cells

Localization of anti-opsin antibodies：Differencein fixation

Immuno-localization of anti-opsin

500 nmMorphogenesis of rod outer segments

Polarity of actin filaments that are found at thetip of the connecting cilium : S1 decoration

200nm

200nm

Electron micrographs of synaptic ribbons

Synaptic ribbons observed fromvarious points of view at mesoscale

300 nm

250 nm0.5 m

Anti-Caveolin 1 antibodies are found on
the synaptic ribbon

Elastic and inelastic scattered electrons

Optical pass way inthe electron
microscope equippedwith γ type energy
filter(co-worked with Tayaand Taniguchi inHitachi)

EELS imaging of Ca in cone synaptic pedicle
High contrast
transmission image

Calculated Ca Image

Merged Image

300nm

Ca is found on the synaptic
ribbon predominantly.

Ca localization determined byEELS imaging

300 nmアクチン細胞骨格の空間構造と空間特異性

Spatial structure and specificity of actin
cytoskeleton

Purpose
We investigate spatial structure of actinfilaments on the membrane and in cytoplasm
by various methods including cryo-electron
microscope, immuno-freeze-etching, scanningelectron microscope and atomic force
microscope.
We are shedding the light on real actin
filaments within cells from various points ofview.

Membrane and cytoplasmic cytoskeleton

Cytoplasmic cytoskeleton

Membrane cytoskeleton
or membrane undercoat

Spatial distribution of membrane actin
filaments

Live cell imaging of GFP-actin in NRK cells

Cytoplasmic surface of cell membrane
covered with many actin filaments

500 nmClathrin and actin filaments

Few membrane
cytoskeletons aredetected even in grazing
thin section,but observed inunroofed freezeetched samples.

Tomography of membrane cytoskeleton

Actin filaments are classified into three types
based on spatial distribution
Actin filaments attached firmly
onto the membrane

Actin filaments loosely attachedon the membrane t

Stress fibers

AFM imaging of membrane cytoskeleton inwaterAFM imaging of membrane cytoskeleton inwaterAFM imaging of clathrin coats in water

Whole cell observation

It is difficult to observe finestructures due to thickcytoplasm.

Cryo-electron micrograph of native cytoskeleton
beneath the cell membraneCryo-electron micrograph of nativecytoskeleton beneath the cell membraneCryo-electron micrograph of native cytoskeleton beneaththe cell membraneSpatial Specificity of Actin Cytoskeletons
found by immuno-labelingProtocol of immuno-freeze-etching

Actin filaments are identified by labeling withcolloidal gold conjugated antibody

Colloidal gold particles are shown here as white dots becauseof reversal contrast.

IQGAP 1

VASP

Cdc 42

Rac 5nm gold / Sla 1 10nm gold

N WASP 10nm gold / IQGAP 1 5 nm gold

Myosin II appeared to be more abundant on stressfibers than the other type of actin filaments on themembrane.

Focal contact related proteins

Integrin b1

High magnification of
boxed area

Focal contact related proteins
Zyxin

Immuno-gold labeling (IQCAP-1) in cryoelectron microscopy

IQCAP-1 is found on actin
filaments located at thecytoplasmic surface of themembrane as a dimer.

200nmDimer

Cryo-electron micrograph showing the dominant area of caveoli
labeled with anti-IQGAP1 immunogold

Summary １
1.

Actin filaments on the membrane were clsaafied into 3 types based on
spatial disposition. First type of actin filaments are tightly bound tomembrane. Second type is the filaments attached loosely to the membrane
and form complicated mesh work at cortical area. Third type is so called
stress fibers.

2.

Binding of effecter proteins indicated spatial specificity. Effecter proteins,
IQGAP1,WASP,VASP bound predominantly on type 2 actin filaments, but
not on type 1. Type 3, stress fiber, also contained their effecter proteins,
but less than Type 2.

1.

Anti-Myosin 2 antibodies was bound on every actin filaments, but a littleon type 1.

1.

After all, type 1 actin filaments was decorated with few actin binding
proteins.

Cytoplasmic and Peri-nuclear
actin filaments

CLSM visualize the cytoskeleton mainly at abottom plane.

Cytoplasmic cytoskeleton observed by extremely
high voltage (1000KV) TEM.

High voltage electron microscopic image of unroofed
pigment epithelial cells.

Myosin II localization in cytoplasmic
cytoskeleton

High resolution SEM image ofcytoplasmic cytoskeleton

Actin filaments in the periphery of nucleus

Many actin filaments are extending from thenuclear envelope

Actin filaments are associated with intermediate filaments inperiphery of the nuclear envelope.

Actin filaments are associated intimately
with intermediate filaments on the nuclear
envelope.

Grazing section of the nucleus labeled with SI

Actin and intermediate filaments originating from
nuclear pores (periphery)

Back scattered electron image showing vimentin
filaments extending from nuclear pores

Anti-vimentin immuno-golds were foundpredominantly in periphery of the nucleus.

Intermediate filaments covering the outer nuclear
envelope were identified as Vimentin.

Anti-Nesprin 1 antibodies are detected
predominantly near nuclear pores.

Summary ２
1.

2.

3.
4.

5.
6.

Cytoplasm was comprised of abundant actin filaments that were not observed
under light microscope. Many actin filaments passed over each other at somepoints and thereby divided the cytoplasm into several domains.
Many actin filaments were attached to nuclear envelope as well. Since S1
decoration showed both pointed and barbed ends clearly in the surface ofnucleus, there seemed to be the terminations and origins of actin filaments on
the nuclear envelope.
Nuclear envelope was covered with thick layer of intermediate filaments,vimentin.
Vimentin filaments extended from nuclear pore like a rosette shape andformed complicated mesh work.
Vimentin filaments associated or covered with almost all actin filaments near
the nuclear envelope.
Nesprin 1 was detected in the periphery of nuclear pores as well.

ご静聴ありがとうございました。

Thank you very much for your attention.