3.Cell Sci, lo, 555-561
(1972)
Printed in Great Britain
LOCALIZATION O F F R E E Z E - F R A C T U R E
PLANES OF YEAST MEMBRANES
F. V. H E R E W A R D
D. H . N O R T H C O T E
Department of Biochemistry, University of Cambridge, Cambridge, C B z I Q W , England
AND
SUMMARY
In freeze-etch replicas the position of the plane of fracture along membranes is controversial;
in an attempt to clarify the situation we have examined drops of yeast suspension frozen at
- 160 OC, fractured and then freeze-substituted with a solution of osmium tetroxide in acetone.
The drops were embedded in Araldite and sections were cut perpendicular to the plane of
fracture to show where the cleavage had occurred.
At the tonoplast the membrane split and the plane of fracture ran between the 2 darkly
staining lines. At the plasmalemma the cleavage plane was not clearly localized. T h e inner half
of the membrane remained with the protoplast and darkly staining material could be seen on
the wall. However, in some places the dark line at the surface of the protoplast could be
resolved into 2 lines. T h e cleavage plane appeared to change at the invaginations of the plasmalemma, where on fracture the whole membrane sometimes remained attached to the wall.
INTRODUCTION
T h e interpretation of ultrastructure revealed by the freeze-etch technique is
restricted by the uncertainty of the plane of fracture along membranes. I t was first
assumed that cleavage had occurred between the membrane and the surrounding
aqueous medium (Moor & Miihlethaler, 1963) but more recent evidence amassed by
Branton and his colleagues shows that membranes can be split at the hydrophobic
interface to reveal their internal structure (Branton, 1966, 1969). Branton's data are
derived largely from the examination of freeze-etch replicas. An alternative approach,
adopted by Bullivant & Weinstein (1969) and Bullivant (1969) and more recently by
Nanninga (1971) has been to thaw, embed and section fixed specimens which had
been fractured in the freeze-etch process. T h e images are difficult to interpret because
as the authors admit, structural changes may occur during warming. T h e method has
only been used to look at one half of the fractured specimen so the fate of the whole
membrane is not unambiguously established.
We have examined fractured drops of yeast suspension frozen for freezesubstitution. Fissures from the plane of fracture show complementary halves of the
membrane and the 2 parts may be observed.
METHOD
The freeze-substitution procedure was a simplified form of that of Van Harreveld (Van
Harreveld & Crowell, 1 9 6 4 ; Hereward & Northcote, 1972). Drops of a suspension of fresh
bakers' yeast (Distillers' Co. Ltd., Great Burgh, Epsom, U.K.) were frozen in freon (Arcton 22,
36
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F. V. Hereward and D. H. Northcote
ICI Ltd., Runcorn, Cheshire, U.K.) cooled by liquid nitrogen. While being frozen the yeast
drops frequently cracked spontaneously, or they could be fractured mechanically while being
held under the freon. The fragments were transferred into a 2 % solution of osmium tetroxide
in dry acetone cooled to —78 °C by solid carbon dioxide. Substitution took 3 days at this
temperature. The specimens and solution were gradually brought to room temperature (1 h at
— 40 C C, 1 h at o °C and 1 h at room temperature). They were then transferred through a
propylene oxide-Araldite series and embedded in Araldite. Sections were cut perpendicular to
the plane of fracture and were stained with a saturated aqueous solution of uranyl acetate for
30 min at 60 °C, then washed and stained with lead citrate for 3 min using the method of Venable
& Coggeshall (1965). They were examined in an AEI EM6B electron microscope at 60 kV.
Freeze-etching was carried out using the Balzers machine fitted with an electron gun (Moor,
Miihlethaler, Waldner & Frey-Wyssling, 1961). After etching for 6 min the surface was
shadowed with tantalum evaporated from a tungsten spigot in the electron gun at 1500-2000 V
and 150 mA (Bachmann, Ambermann & Zinbsheim, 1969).
RESULTS
Fractures were frequently observed at the plasmalemma (Figs. 6-8 and 10), tonoplast (Figs. 1-5) and nuclear membranes. The tonoplast fractures were the most easily
interpreted and showed that the membrane had split. In a fractured cell the intact
tonoplast was resolved into 2 darkly staining components (Figs. 1-5). One of these
was continuous with the membrane on the fractured face which always appeared as a
single darkly staining line (Figs. 1-5). Where cracks through the cell had occurred at
the tonoplast each of the complementary fractured surfaces was limited by a single
line (Figs. 1, 2, 5). At the base of the tonoplast in freeze-etched yeast there was some
indication of a ledge since deep etching had exposed the outer face of the membrane.
The intact plasmalemma in the freeze-substituted cell appeared as one well defined
line at the edge of the cytoplasm and a second darkly staining band against the wall
separated from the first by a clear space (Fig. 7). These 2 dark lines each had a well
defined edge at the clear space which separated them. We were not able to locate this
space in the fractured cells because although a single darkly staining line separated
with the wall, the complementary surface of the cytoplasm was sometimes limited by
a double line, the inner portion of which corresponded to the inner half of the
membrane in unfractured cells (Figs. 6-8, 10). Outside this well-defined inner
component of the plasmalemma and separated from it by a diffuse unstained lumen, a
thinner line could sometimes be seen (Figs. 6-8, 10). The lumen, seen in fractured
cells, was narrower than that separating the 2 dark halves of the membrane in intact
cells. In cells with small fissures the thinner line sometimes appeared but could not be
seen to be continuous with either of the 2 dark lines (Fig. 7). Where the fracture
which split the wall from the cell had passed across the wall, stain had been deposited
at the edges of the cross-fracture (Figs. 6, 7 and 10).
The plane of cleavage could change where the plasmalemma formed invaginations.
Here the fracture usually resulted in the 2 halves of the plasmalemma in the intact
cell having adhered to the wall side of the cleavage plane, and at the complementary'
points on the cytoplasmic side a very diffuse edge was seen (Figs. 6-8). When these
invaginations were examined in freeze-etched cells (Fig. 9), a ridge could often be
seen where the membrane had fractured away from the cytoplasm to follow the
contours of the wall.
Freeze-fracture planes
557
DISCUSSION
In yeast frozen in liquid freon at — 160 °C fractures occur spontaneously or may be
induced. We consider that the planes of cleavage are the same as those found in
freeze-etched specimens fractured at — 100 °C by a knife cooled to - 180 °C.
Studies on the fracture planes of the tonoplast show that this membrane is split.
The bilamellar membrane can be seen in the cytoplasm, one half continuous with a
single darkly staining line on the fractured face; fissures passing down into the
fractured drop show complementary surfaces in which each fractured face is limited
by one half of the membrane.
At the plasmalemma the situation is more complex. Although on fracture darkly
staining lines separate with the wall and protoplast, these are not necessarily the two
halves of the membrane because a third layer can sometimes be resolved on the
outermost surface of the protoplast, separated from the inner half of the plasmalemma
by a diffuse, clear lumen. This could represent the outer half of the membrane, in
which case the dark line on the wall results either from the staining of an inner layer
of wall material (Matile, Moor & Robinow, 1969) which is continuous with the outer
material of the plasmalemma in the intact cell and so not distinguished from it, or
from the deposition of stain on the freshly exposed surface as occurs at cross-fractures
through the wall. If either of these explanations is correct then the plane of fracture
would be between the wall and the plasmalemma. Alternatively the indistinct third
line could have resulted from non-specific adsorption of stain in which case the
membrane would have been split. However no such non-specific adsorption has
occurred on the exposed surface of the fractured tonoplast so the convex plasmalemma
surface has different staining properties. Branton has examined the fractured faces of
freeze-etched yeast (Branton & Southworth, 1967). He was unable to find evidence
that the membrane had split and admits that the exposed face may be the true
plasmalemma surface. Staehelin (1968) has good evidence that the plasmalemma of
Cyanidium caldarium is unsplit.
At the invaginations of the plasmalemma the plane of fracture can change. Both
halves of the membrane can remain with the wall. The examination of complementary
freeze-etch replicas has shown for all membranes studied that there is a unique plane
of fracture (Chalcroft & Bullivant, 1970; Sleytr, 19700,6; Wehrli, Miihlethaler &
Moor, 1970; Nanninga, 1971). A single plane would be expected for the tonoplast as
it has split. The behaviour on fracture of the plasmalemma changes where localized
differentiation has occurred to give invaginations. Thus in different situations a single
membrane need not have a unique fracture plane. Changes in membrane conformation
or function have produced differences in the forces between the membrane and its
environment. It is therefore unjustifiable to generalize from one membrane to another
and the unambiguous evidence at the tonoplast is irrelevant to the plasmalemma. The
unique plane of fracture of the plasmalemma (Sleytr, 1970a, b), excluding the
invaginations, may occur within the membrane or it may pass between the wall
and the membrane. Either of these fracture planes would result in surfaces which
would not be expected to etch as they expose either the internal surfaces of the
36-2
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F. V. Hereward and D. H. Northcote
membrane or the inner surface of the wall and the outer surface of the plasmalemma.
F.V. H. thanks the Science Research Council for a grant during the tenure of which this
research was carried out.
REFERENCES
BACHMANN, L., AMBERMANN, R. & ZINBSHEIM, H. P. (1969). Ultra-shadowing in freeze-
etching. J. Cell Biol. 43, 8 A.
BRANTON, D. (1966). Fracture faces of frozen membranes. Proc. natn. Acad. Set. U.S.A. 55,
1048-1056.
BRANTON, D. (1969). Membrane structure. A. Rev. PL Physiol. 20, 209-238.
BRANTON, D. & SOUTHWORTH, D. (1967). Fracture faces of frozen Cldorella and Saccliaroviyces
cells. Expl Cell Res. 47, 648-653.
BULLIVANT, S. (1969). Freeze-fracturing of biological materials. Micron 1, 46—51.
BULLIVANT, S. & WEINSTEIN, R. S. (1969). A thin section study of the path of fracture planes
along frozen membranes. Anat. Rec. 163, 296.
CHALCROFT, J. P. & BULLIVANT, S. (1970). An interpretation of liver cell membrane and
junction structure based on observation of freeze-fracture replicas of both sides of the fracture.
J. Cell Biol. 47, 49-60.
HEREWARD, F. V. & NORTHCOTE, D. H. (1972). A simple freeze-substitution method for the
study of ultrastructure of plant tissues. Expl Cell Res. 70, 73-80.
MATILE, P., MOOR, H. & ROBINOW, C. F. (1969). Yeast cytology. In The Yeasts, vol. 1 (ed.
A. H. Rose & J. S. Harrison), pp. 219-302. New York and London: Academic Press.
MOOR, H. & MOHLETHALER, K. (1963). Fine structure in frozen-etched yeast cells. J. Cell Biol.
17, 609-628.
MOOR, H., MOHLETHALER, K., WALDNER, H. & FREY-WYSSLING, A. (1961). A new freezing-
ultramicrotome. J. biophys. biocheni. Cytol. 10, 1-13.
NANNINGA, N. (1971). Uniqueness and location of the fracture plane in the plasma membrane
of Bacillus subtilis.J. Cell Biol. 49, 564-570.
SLEYTR, U. B. (1970a). Die Gefrieratzung korrespondierender Bruchhalften: ein neuer Weg
zur Aufklarung von Membranstrukturen. Protoplasma 70, 101-117.
SLEYTR, U. B. (19706). Fracture faces in intact cells and protoplasts of Bacillus stearothennophilus. A study by conventional freeze-etching and freeze-etching of corresponding fracture
moieties. Protoplasma 71, 295-312.
STAEHELIN, L. A. (1968). Ultrastructural changes of the plasmalemma and the cell wall during
the life cycle of Cyanidium caldarium. Proc. R. Soc. B 171, 249-259.
VAN HARREVELD, A. & CROWELL, J. (1964). Electron microscopy after rapid freezing on a metal
surface and substitution fixation. Anat. Rec. 149, 381—385.
VENABLE, J. H. & COGGESHALL, R. (1965). A simplified lead citrate stain for use in electron
microscopy. J. Cell Biol. 25, 407-408.
WEHRLI, E., MOHLETHALER, K. & MOOR, H. (1970). Membrane structure as seen with a
double replica method for freeze fracturing. Expl Cell Res. 59, 336—339.
(Received 2 September 1971)
ABBREVIATIONS ON PLATES
c
cf
/
i
r ridge
cytoplasm
cross-fracture
t tonoplast
fracture
v vacuole
invagination
tv wall
The scale mark on the figures is 500 nm.
Freeze-fracture planes
Figs. 1-5. For legend see p. 560.
559
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F. V. Hereward and D. H. Northcote
Fig. 1. A fissure has passed through the cytoplasm and over the vacuole but the 2
complementary halves have remained closely apposed. Dark lines limit the fractured
halves, these are further apart than in the unfractured region, x 55000.
Fig. 2. A fissure has occurred over the vacuole. The inner line of the bilamellar
membrane can be seen to be continuous with that at the edge of the fracture face. On
the complementary half of the fractured cell there is some indication of the outer half
of the membrane, x 61000.
Fig. 3. A fracture has passed through a cell and across the vacuole. The outer half of
the membrane persists for a short distance along the fracture face, the inner half has
fractured away. At the position of arrow 1 a partial separation has occurred and the
distance between the halves of the membrane is greater than that at the position of
arrow 2. x 77000.
Fig. 4. A fissure through the cell has passed over the tonoplast. The inner half of the
membrane is continuous with the membrane on the fractured face. The outer half,
which is clearly visible in the intact parts of the cell, has fractured away, x 84000.
Fig. 5. A fissure has passed through the cytoplasm and over the tonoplast. The
intact membrane is resolved into 2 darkly staining lines, each of the fractured faces is
limited by 1 of these lines, x 68000.
Fig. 6. The wall has fractured away from the cytoplasm. The exposed surfaces and
the cross-fractured wall are each limited by a dark line but in some places a double line
can be seen on the surface of the cell (arrow). At the invaginations of the plasmalemma
the membrane has adhered to the wall and there is a very diffuse edge at the complementary points on the cell, x 100000.
Fig. 7. A small fissure into a cell has occurred. Stain has been deposited on the crossfractured edges of the wall. The wall has separated slightly from the protoplast. A
faint line appears between the dark lines limiting the cytoplasm and the wall (arrow).
The membrane adheres to the wall at the invaginations. x 104000.
Fig. 8. The convex surface of the fractured yeast is clearly limited by a single darkly
staining line, but there is some indication of a double line at one point (arrow).
There is a diffuse edge at the invaginations. x 85000.
Fig. 9. A freeze-etched yeast. Ridges can be seen to run along the invaginations where
part of the membrane has fractured away. Encircled arrow indicates the direction of
the tantalum shadow, x 50000.
Fig. 10. A fracture has passed between the cell and the wall. The exposed surfaces and
the cross-fractured edges of the wall are each limited by a darkly staining line but that
on the cell can be resolved into 2 lines in some places (arrow), x 78000.
Freeze-fracture planes
0