Vol. 181, No. 2, 1991 December 16, 1991
BIOCHEMICAL
SCANNING
MICROSCOPY
M.
TUNNELING
Gaczynskaf
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 600-603
OF HUMAN ERYTHROCYTE
M. Chwialkowski: W. Olejniczakf and G. Bartoszf
MEMBRANES
S. Wojczuk:
*Department of Solid State Physics, Institute of Physics and #Laboratory of Biophysics of Development and Aging, Department of Biophysics, University of Lodz, Banacha 12-16, 90-237 Lodz,Poland Received
October
11,
1991
Images of surfaces of human erythrocyte ghosts, lecithin liposomes, spectrin, erythrocyte membrane skeleton, concanavalin A and concanavalin A - decorated erythrocyte ghosts were obtained by scanning tunneling microscopy. The dimensions and surface topography of some membrane structures are described and discussed. 0 1991 Academic Press,
*nc.
STM is
a promising
account
of
natural
conditions
especially
and of
MATERIALS
surface
imaging
Research for
the
analysis living
of
surface
biomembranes.
has been
we presenr
some their
of
[1,2].
information report
for
possibility
interesting
limited this
its
technique
extracted first
matter
observation
under
topography
However, from
in biology
till
on almost
may be now only
such
studies
of
erythrocyte
[3,4].
In
ghosts
structures.
AND METHODS
in the constant height mode [5]. The Our STM operated in air, current of mean voltage of 1.2 V - 1.5 V was used and tunneling value of 1.5 nA was measured. Samples (deposited on a vacuum sublimated gold film) were negative with respect to the tip. Human erythrocyte membranes Surfaces of 250-2500 nm2 were scanned. Crude spectrin extract and were prepared by hypotonic hemolysis [6]. erythrocyte membrane skeleton were obtained according to Horne-et al. [7]. Lecithin (Sigma) multilamellar liposomes were prepared by shaking the lecithin film with 5 mmol/l sodium phosphate buffer, pH 7.4. ConA (Research Products: 500 ug/ml) in phosphate buffer) was incubated (lh, room temperature) with ghosts (500 ug protein/ml buffer). Unbound ConA was washed away. The preparations were diluted with distilled water to about 5 ug of membrane protein or lecithin per ml (ConA: 1 rig/ml). A droplet of a preparation was pipetted on gold film and air-dried. At least 10 images of various pOSitiOnS for at least 2 droplets of each sample were analysed. 0006-291x/91 $1.50 Copyright All rights
6 1991 by Academic Press, Inc. of reproduction in any form reserved.
600
Vol.
BIOCHEMICAL
181, No. 2, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS Clear
and reproducible
ghosts,
lecithin
images
liposomes,
ConA and ConA-decorated all
the
samples
folds
(Fig.
background
with
in good
molecules Long
(diameter:
about
1,3).
Some
structures,
In
more
of
the
a plain
of
complicated
against
for
molecules.
filaments skeleton
on which
small
regular
this
were of
spectrin
nm corresponding
were
film
Plain observed
these
round
ConA molecules linked
areas
[8-lo].
apparently
with
together
A characteristic was worth
to
images
a net
to
subunits were
observed
by small O1wavy@'
note of
(Fig.
3a,b).
filaments
a
-5
01
-
1Onm
1. STM images Data subjected Four hilly structures Con A tetramer.
%-
Fig. 2. membrane filtration Extending
nm
02 of Con A (a) and Con to median filtration in the lower part
A-decorated erythrocyte and reversion versus of (a) correspond to
STM images of liposome surface (a) and of the red cell surface (b). The pictures were obtained after surface of data and reversion of data versus the background. structures correspond to membrane proteins.
601
for
and ConA
respectively
to
skeleton,
applied.
current
structures,
threads
membrane
with
samples
and tetramers
actin
spectrin
image
and dimensions
5 nm) corresponding spectrin
The gold
foreseen
3-4
erythrocyte membrane
observed
biological
skeleton
molecules
probably
structure
gave
those
of diameter
spectrin
obtained.
Shapes
with
and membrane
were
low tunneling
l-3).
agreement
elongated
(Fig.
yielding
(Fig.
filaments
the
of human
erythrocyte
structures
from
areas
preparations
were
the
originated
surfaces all
deposited
so all
surfaces
spectrin, ghosts
were
la)
of
membranes the background. subunits of the
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
-20
RESEARCH COMMUNICATIONS
nm
Fi . 3. Images and surface topography of human erythrocyte spectrin a,b and cytoskeleton (c,d). STM data subjected to median filtration; (a) and (d) also reverted with respect to the background. Relative values of the tunneling current are on the scales in (b) and (c). The same fragments of samples are presented on (a-b) and (c-d) pictures. A wavy fine structure of spectrin filaments can be seen in (a).
F-J-
(spectrin)
and non-filament
Scanning
of
whereas
the
presence
of
2b).
ConA-treated
For
significantly for
ghosts
areas
liposomes
gave
topography small
structures
reacted
can be ascribed
of plain
map of erythrocyte
(diameter
higher:
images
were
of about
membranes
the
4+1 per
250 nm
with
proteins
ghosts
amount
jutting
surface. 602
for
(Fig.
surfaces
of
surface
n=4)
(Fig. out
the
2a) showed
structures
these
control
3c,d).
(Fig.
2-3 nm) round
ConA (mean+SE; to
observed
the (Fig.
structures ghosts lb,
3b).
external
was
and 17+2 These membrane
Vol.
BIOCHEMICAL
181, No. 2, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
DISCUSSION In
all
cases
'lchannelsll images
studied in
units
produced
conductivity which It
of
tunneling
the
sample
and the
effects
on the
suggested
that
order
of magnitude
for
poorly
conducting
layer
under
the
conducting
the
every
thickness value
of the
of the
[ll]. in this
structures
diminished
the
skeleton
observed
agreement
with
in
those
some room of
for
structures this
study
obtained
imaging
of
current not
(Fig.
using
a confidence
thickness
electron
with
supramolecular
respect
of external
study
to in
the
poorly
any fixation
reasonable
microscopy to
also
[2].
subjected 3) are
by an
Probably
values
layer current.
decreases
DNA molecule employed
sample
tunneling
current
angstrom
surface
the
of the of the
tunneling
or
However,
of two factors:
conditions
procedure
possibilities
result
as ~~holestl
values.
measurement
The membrane
gives
current
the
opposite
has been
were visualised
by STM are of
have
proteins
[9,10]
which
current
structures.
ACKNOWLEDGMENTS The authors would like to thank Prof. for interest. This work was performed Project CPBP 01.08 "Surface Physics".
L. Wojtczak within the
and Prof. framework
W. Leyko of the
REFERENCES 1. Feng,L., (1989) Scanning Microscopy 3, Andrade,J.D., Hu,C.Z. 399-410. 2. Baro,A.M., Miranda, R., Alaman,J., Garcia,N., Binning,G., Rohrer,H., Gerber,C., Carrascosa,J.L. (1985) Nature 315, 253-254. 3. Zasadzinski,J.A.N., Schneir,J., Gurley,J., Elings,V., Hansma, P.K. (1988) Science 239, 1013-1015. 4. Ruppersberg,J.P., Horber,J.K.H., Gerber,C., Binnig G. (1990) FEBS Lett. 257, 460-464. 5. Olejniczak,W. et al. (1990) Materials of International Conference STM'9O/NANO I, Baltimore, in press. 6. Gaczynska,M., Bartosz,G. (1989) Int. J. Biochem. 21, 1383-1385. 7. Horne,W.C., Leto,T.L., Anderson,R.A. (1989) Meth.Enzymol. 173, 380-392. 8. Horber,J.K.H., Lang,A., Hansch,T.W., Heckl,W.M., Mohwald, H. (1988) Chem. Phys. Lett. 145, 151-158. 9. McGough,A.M., Josephs,R. (1990) Proc. Nat. Acad. Sci. USA 87, 208-212. 10. Shen,B.W., Josephs,R., Steck,T.L. (1984) J. Cell Biol. 99, 810-821. 11. Keller,D., Bustamante,C., Keller,R.W. (1989) Proc. Nat. Acad. Sci. USA 86, 5356-5360. 603
BIOCHEMICAL
SCANNING
MICROSCOPY
M.
TUNNELING
Gaczynskaf
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 600-603
OF HUMAN ERYTHROCYTE
M. Chwialkowski: W. Olejniczakf and G. Bartoszf
MEMBRANES
S. Wojczuk:
*Department of Solid State Physics, Institute of Physics and #Laboratory of Biophysics of Development and Aging, Department of Biophysics, University of Lodz, Banacha 12-16, 90-237 Lodz,Poland Received
October
11,
1991
Images of surfaces of human erythrocyte ghosts, lecithin liposomes, spectrin, erythrocyte membrane skeleton, concanavalin A and concanavalin A - decorated erythrocyte ghosts were obtained by scanning tunneling microscopy. The dimensions and surface topography of some membrane structures are described and discussed. 0 1991 Academic Press,
*nc.
STM is
a promising
account
of
natural
conditions
especially
and of
MATERIALS
surface
imaging
Research for
the
analysis living
of
surface
biomembranes.
has been
we presenr
some their
of
[1,2].
information report
for
possibility
interesting
limited this
its
technique
extracted first
matter
observation
under
topography
However, from
in biology
till
on almost
may be now only
such
studies
of
erythrocyte
[3,4].
In
ghosts
structures.
AND METHODS
in the constant height mode [5]. The Our STM operated in air, current of mean voltage of 1.2 V - 1.5 V was used and tunneling value of 1.5 nA was measured. Samples (deposited on a vacuum sublimated gold film) were negative with respect to the tip. Human erythrocyte membranes Surfaces of 250-2500 nm2 were scanned. Crude spectrin extract and were prepared by hypotonic hemolysis [6]. erythrocyte membrane skeleton were obtained according to Horne-et al. [7]. Lecithin (Sigma) multilamellar liposomes were prepared by shaking the lecithin film with 5 mmol/l sodium phosphate buffer, pH 7.4. ConA (Research Products: 500 ug/ml) in phosphate buffer) was incubated (lh, room temperature) with ghosts (500 ug protein/ml buffer). Unbound ConA was washed away. The preparations were diluted with distilled water to about 5 ug of membrane protein or lecithin per ml (ConA: 1 rig/ml). A droplet of a preparation was pipetted on gold film and air-dried. At least 10 images of various pOSitiOnS for at least 2 droplets of each sample were analysed. 0006-291x/91 $1.50 Copyright All rights
6 1991 by Academic Press, Inc. of reproduction in any form reserved.
600
Vol.
BIOCHEMICAL
181, No. 2, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS Clear
and reproducible
ghosts,
lecithin
images
liposomes,
ConA and ConA-decorated all
the
samples
folds
(Fig.
background
with
in good
molecules Long
(diameter:
about
1,3).
Some
structures,
In
more
of
the
a plain
of
complicated
against
for
molecules.
filaments skeleton
on which
small
regular
this
were of
spectrin
nm corresponding
were
film
Plain observed
these
round
ConA molecules linked
areas
[8-lo].
apparently
with
together
A characteristic was worth
to
images
a net
to
subunits were
observed
by small O1wavy@'
note of
(Fig.
3a,b).
filaments
a
-5
01
-
1Onm
1. STM images Data subjected Four hilly structures Con A tetramer.
%-
Fig. 2. membrane filtration Extending
nm
02 of Con A (a) and Con to median filtration in the lower part
A-decorated erythrocyte and reversion versus of (a) correspond to
STM images of liposome surface (a) and of the red cell surface (b). The pictures were obtained after surface of data and reversion of data versus the background. structures correspond to membrane proteins.
601
for
and ConA
respectively
to
skeleton,
applied.
current
structures,
threads
membrane
with
samples
and tetramers
actin
spectrin
image
and dimensions
5 nm) corresponding spectrin
The gold
foreseen
3-4
erythrocyte membrane
observed
biological
skeleton
molecules
probably
structure
gave
those
of diameter
spectrin
obtained.
Shapes
with
and membrane
were
low tunneling
l-3).
agreement
elongated
(Fig.
yielding
(Fig.
filaments
the
of human
erythrocyte
structures
from
areas
preparations
were
the
originated
surfaces all
deposited
so all
surfaces
spectrin, ghosts
were
la)
of
membranes the background. subunits of the
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
-20
RESEARCH COMMUNICATIONS
nm
Fi . 3. Images and surface topography of human erythrocyte spectrin a,b and cytoskeleton (c,d). STM data subjected to median filtration; (a) and (d) also reverted with respect to the background. Relative values of the tunneling current are on the scales in (b) and (c). The same fragments of samples are presented on (a-b) and (c-d) pictures. A wavy fine structure of spectrin filaments can be seen in (a).
F-J-
(spectrin)
and non-filament
Scanning
of
whereas
the
presence
of
2b).
ConA-treated
For
significantly for
ghosts
areas
liposomes
gave
topography small
structures
reacted
can be ascribed
of plain
map of erythrocyte
(diameter
higher:
images
were
of about
membranes
the
4+1 per
250 nm
with
proteins
ghosts
amount
jutting
surface. 602
for
(Fig.
surfaces
of
surface
n=4)
(Fig. out
the
2a) showed
structures
these
control
3c,d).
(Fig.
2-3 nm) round
ConA (mean+SE; to
observed
the (Fig.
structures ghosts lb,
3b).
external
was
and 17+2 These membrane
Vol.
BIOCHEMICAL
181, No. 2, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
DISCUSSION In
all
cases
'lchannelsll images
studied in
units
produced
conductivity which It
of
tunneling
the
sample
and the
effects
on the
suggested
that
order
of magnitude
for
poorly
conducting
layer
under
the
conducting
the
every
thickness value
of the
of the
[ll]. in this
structures
diminished
the
skeleton
observed
agreement
with
in
those
some room of
for
structures this
study
obtained
imaging
of
current not
(Fig.
using
a confidence
thickness
electron
with
supramolecular
respect
of external
study
to in
the
poorly
any fixation
reasonable
microscopy to
also
[2].
subjected 3) are
by an
Probably
values
layer current.
decreases
DNA molecule employed
sample
tunneling
current
angstrom
surface
the
of the of the
tunneling
or
However,
of two factors:
conditions
procedure
possibilities
result
as ~~holestl
values.
measurement
The membrane
gives
current
the
opposite
has been
were visualised
by STM are of
have
proteins
[9,10]
which
current
structures.
ACKNOWLEDGMENTS The authors would like to thank Prof. for interest. This work was performed Project CPBP 01.08 "Surface Physics".
L. Wojtczak within the
and Prof. framework
W. Leyko of the
REFERENCES 1. Feng,L., (1989) Scanning Microscopy 3, Andrade,J.D., Hu,C.Z. 399-410. 2. Baro,A.M., Miranda, R., Alaman,J., Garcia,N., Binning,G., Rohrer,H., Gerber,C., Carrascosa,J.L. (1985) Nature 315, 253-254. 3. Zasadzinski,J.A.N., Schneir,J., Gurley,J., Elings,V., Hansma, P.K. (1988) Science 239, 1013-1015. 4. Ruppersberg,J.P., Horber,J.K.H., Gerber,C., Binnig G. (1990) FEBS Lett. 257, 460-464. 5. Olejniczak,W. et al. (1990) Materials of International Conference STM'9O/NANO I, Baltimore, in press. 6. Gaczynska,M., Bartosz,G. (1989) Int. J. Biochem. 21, 1383-1385. 7. Horne,W.C., Leto,T.L., Anderson,R.A. (1989) Meth.Enzymol. 173, 380-392. 8. Horber,J.K.H., Lang,A., Hansch,T.W., Heckl,W.M., Mohwald, H. (1988) Chem. Phys. Lett. 145, 151-158. 9. McGough,A.M., Josephs,R. (1990) Proc. Nat. Acad. Sci. USA 87, 208-212. 10. Shen,B.W., Josephs,R., Steck,T.L. (1984) J. Cell Biol. 99, 810-821. 11. Keller,D., Bustamante,C., Keller,R.W. (1989) Proc. Nat. Acad. Sci. USA 86, 5356-5360. 603
