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OCR for page 155
MEMBRANE MODELS: E\tOL{JTION FROM IRE EWID—MOSAIC STANDBY
ROY HAMMERS TEDT
INTRODUCTION
Postulation of the fluid-mosaic model for membrane structure by
Singer and Nicholson (1972) represented a thoughtful and reasoned
synthesis of information obtained from observations extending over loo
years. These many contributions, summarized by Robertson (1981), began
with the implicit recognition by Schleiden and Schwann that a membrane
structure Rust bound the cell, and includes contributions from
physicists, chemists and morphologists. Other key elements were: use of
evolving techniques to highlight new aspects of the complex structure
leg., electron microscopy); careful quantitative analyses of the sizes
and numbers of the postulated elements of the membrane (protein, sterol
and phospholipids); and characterization of the physical features of
membranes, either as isolated or after resolution into individual
components. The resulting model has served as an framework upon which a
host of experiments have been conducted to further illuminate the role of
the interface between the cell interior and its environment or between
compartments within the cell. Since the presentation of the fluid-mosaic
model, continuous application of pre-existing plus new techniques has
resulted in additional quantitative details that must be integrated into
the model. Careful rereading of this classic paper, as opposed to simple
inspection of the much imitated schematic Figures (see Figure 1, left
panel), reveals that the authors clearly understood the limitations to
their proposal and anticipated some of the modifications proposed later
by others (Figure 1, right panel).
Figure 1. Schematic representation of bilayer membrane. The progressive
evolution of our concepts of the structure of the lipid bilayer is
illustrated by the initial presentation (Singer and Niclolson, 1972) on
the left and an "updated" version (Cullis and Hope, 1985) on the right.
Figures reprinted with permission of the publishers.
The fluid mosaic model retained earlier concepts leg., a lipid
bilayer by Danielle and Davson) while providing a new way of interpreting
the distribution of protein. Proteins were considered to penetrate into
the lipid ~ in an iceberg fashion) leaving most of the inner and outer
surfaces as naked lipid. This mosaic pattern provided a portion of the
name. The second aspect, fluidity, encompassed the observations of Frye
and Edidin (1970) on the ability of fluorescent dyes to move freely in
the membrane. These two aspects (fluidity and mosaic) remain dominant
features of the evolving membrane model.
— 155 —
OCR for page 156
Contributions of others will be reviewed herein to illustrate: an
increased awareness of the complexity of the outer surface of the plasma
membrane; the asymmetric distribution of lipid components across the
bilayer, and the effect that these distributions have on the physical
properties of the membrane; and the lateral organization (domain
structure) of the membrane. These revisions are especially important for
understanding of membrane events on the surface, or plasma, membrane.
Final comment" will be directed to the design and interpretation of
experiments related to reproduction and will emphasize the dynamics of
time frame and spatial relationships.
COMPLEXITY OF THE CELL SURFACE
The concept of integral ( intrinsic) and peripheral
· . . .
(extrinsic)
proteins found in the original fluid-mosaic model illustrates an
awareness that the external face of the membrane was complex, and the
differential staining of the outer surface of cells (Revel, et al, 1960)
provided strong evidence for the specialized distribution of carbohydrate
on the cell surface. The growing array of biophysical techniques
available to study the properties of cell surfaces (pp 31-48 of Rao,
1987 ~ provide a large body of evidence that the surface membranes of
cells differ, and that ~layers" of unique molecules extend from the
bilayer into the surrounding media. Molecules in this succession of
layers (Figure 2) "shield" the bilayer from the surrounding solution.
Exposure to external phospholipases resulted in greatly different extents
of hydrolysis of phospholipids of erythrocytes and erythrocyte ghosts
(Zwall and Roelofson (1976) and Ottolenghe (1973) ) . This was because of
shielding, as illustrated in Figure 2.
Figure Z. Schematic representation of the layers found on the cell
surface. This scale drawing illustrates the types of molecules, and
.
their spacla1 relatlonsnlps, Anal can exist on one surface or a cell.
The frequency of appearance of the proteins that extend from the bilayer
to form the glycocalyx or are adsorbed is unknown, and probably differs
among cell types. The relative sizes of soluble components of molecular
weights of 200 and 60,000 are illustrated. Adapted from Susko-Parrish,
et al., 1985.
156 -
OCR for page 157
PUS'M
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OCR for page 158
This succession of layers undoubtedly exists on the surface of
many-to-most cells, but the distribution of the components and their
density on the surface is difficult to establish. Adsorbed protein
senre'; as ~ general descriptor for many of these macromolecules, and the
necessity of washing cells before analysis wil 1 remove loosely bound
components before the analysis begins. These complicating factors make
interpretation of data obtained from such suspensions very dif f icult .
Hormone-receptor binding analyzes often reveal alterations in the
number of receptor site" as a function of physiological state. In many
cases, but certainly not all, subsequent analyses established that
biosynthetic and degradative processes resulted in removal of (or
addition to) receptors from the bilayer. Inspection of Figure 2, which
is drawn to scale, shows how access of an extracellular ligand (the large
sphere represents ~ molecule of about 60,000 daltons) to the bilayer is
dependent on the disposition of other surface components exterior to the
bilayer. In contrast, the approach of a small molecule (eg. glucose;
"mall sphere) is less likely to be impeded e Such hinderances often are
not considered in the analysis of surface binding phenomena.
If the ligand for study has only one class of binding sites, the
binding properties can be accurately assessed. Unfortunately, multiple
classes of binding sites introduce complexities unrecognized by most
investigators. The reader should consult the articles of Klotz (1982,
1983) for examples and details. One example of multiple sites is the
analysis of lectin binding mites on the sperm surface. Susko-Parish et
al. (1985) tested the use of iodinated lectin and classical Skatchard
analysis for this purpose; no reliable quantitative data were obtained.
Subsequently, Magargee et al (1988) utilized flow cytometry, under
carefully standardized conditions, for cell-by-cell analysis of the same
phenomenon and were able to guantitate relative binding to the sperm
surface for a variety of lectins.
ASYMMETRY OF CELL SURFACES
This concept also was considered in the original fluid-mosaic model,
where the pictorial displays clearly indicate that components in the
membrane were unequally distributed relative to the exterior and interior
faces (Figure 11. Further, based on staining properties mentioned
previously, carbohydrate components (eg., gangliosides) were selectively
enriched on the exterior face of the bilayer. The major contribution of
the past decade was the delineation of the orientation of the
phospholipids of the bilayer. These concepts are lucidly described in a
review by Cullis and Hope (1985) and the texts by Houslay and Stanley
(1982) and Jain (19873; the reader is encouraged to study these
contributions for the primary references and exposure to the limitations
to the general statements presented below.
t
Analysis of the lipid components in membranes involves: isolation
of the cell (s) of interest free of ~significant" contaminants; removal
and purification of membranes, including providing proof of their point
of origin in the cell; extraction of the lipids from the recovered
membrane vesicles; and analysis of the lipid classes. Methods to
accomplish these interdependent steps have been developed, and the
— 158 —
OCR for page 159
resulting data base describes the mayor lipids for a variety of cell and
membrane types. The large variety specific molecular forms leg.,
phospholipid types with a variety of acyl substituents) has been
bewildering, in that no general structural solution to the common
biologic.] problem of assembly of a stable bilayer was apparent. Major
contributions of the last decade have been in identifying common features
of these components to allow general statements about membrane structure.
The phospholipid asymmetry of plasma membranes for five cell types
is presented in Figure 3. For a membrane of the dimensions of ~ cell, a
stable bilayer will have the phospholipids equally divided between the
outer and inner surfaces (monolayers or leaflets). However, the
individual phospholipid types need not be equally distributed toward the
inner and outer monolayers. This certainly is true for the plasma
membrane, where phosphatidy~choline (PC) and sphingomyelin (SPM) are
preferentially oriented ~out" with phosphatidylethanolamine (PE),
phosphatidylserine (PS) and phosphatidylinositol (PI) preferentially
oriented gins. Since the phospholipid classes differ in the general
types of acyl substituents, with PC and SPM having the more saturated
members and PE, PS and PI having the more unsaturated components, this
asymmetry in lipid by head group also introduces asymmetry in the
hydrocarbon portion of the bilayer. To a first approximation, this
asymmetry between outer and inner leaflets could result in differences in
flexibility gradient (freedom of motion proceeding from the bilayer face
into the interior) for the exterior and interior faces. Model system
studies also establish that cholesterol (CHOL) preferentially interacts
with SPM and PC relative to PE, PS and PI, thereby introducing the
possibility of a asymmetric distribution of the sterols. Definitive
proof of the asymmetric distribution of cholesterol in cells has been
difficult because of a dearth of methods to establish sterol
localization, but the data for model systems argue strongly for this
feature.
Figure 3: Phospho] ipid distributions for surface membranes . The
percentage of the phosphor ipids that are oriented to the outer vs the
inner faces of the plasma membrane are shown for : A, human erythrocyte;
B. rat liver sinusoidal surface; C, rat liver continuous surface; D, pig
platelet; and E, BHK-21 cells. Abbreviations for the lipids are
presented in the text. Taken from Cullis and Hope (1985) with permission
of the publisher. ~: ;1 _ _
- 159 -
OCR for page 160
Detailed studies of model lipid dispersions have proven most useful
to the resolution of these difficulties, in that precise spectroscopic
and microscopic methods can be applied to the analysis of lipid assembly
into phases. A clear representation of several possibilities (Figure 4
considers two general cases . First and most predominant, a situation
where molecules cannot exhibit total freedom of motion in all dimensions
is classified as an anisotropic systems (bilayer and hexagonal (Hal) ~ .
The second, where complete freedom of motion can occur, is an isotropic
mode. Specific examples include a bilayer (orientation of headgroup out,
and respective acyl side chains toward each other and intercalated), the
hexagonal phase (polar head group'; in and acyl side chains out) and any
of 'several orientations (vesicles, micellar, etc. ~ where ache members
have isotropic motion. Each is recognizable by virtue of its nuclear
magnetic resonance (~) spectroscopic features and the images gathered
by freeze fracture electron microscopy (EM) analysis. Detailed studies
of many types of lipids have established that each has a preference for
the phase it will adopt when placed in an aqueous environment. Some
lipids (PC, SPM, PS (at pH > 3 ), PI) prefer the bilayer while others (PE ,
PS flat pH ~ 3 ), CHOL) prefer the hexTI orientation. If a membrane was
composed of only one-lipid, reasonably accurate predictions of the phase
of the membrane formed could be made. Even then, predictive ability is
limited as evident from observations that addition of secondary
components ~ cholesterol or long-chain unsaturated fatty acids ~ to l ipids
preferring the bilayer form will induce a transition to a hexagonal
orientation.
[inure 4. Unique NMR spectra and freeze-fracture patterns for various
phospholipid phases. Preparation of lipid dispersions to yield putative
bilayer, hexanonal and isotropic phases, followed by analyses by NMR
(center panel) and electron microscopy (right panel) established the
unique features of each phase. Taken from Cullis and Hope (1988) with
permission of the publisher.
P. ',l~li~ - _ C. ala. 81, '4~' a_C~
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~3
- 160 -
OCR for page 161
Considerable attention has been given to the changes in the degree
of molecular ordering observed when temperature of the lipid phase is
altered. At low temperatures the bilayer exists in a crystalline state,
where the acyl chains are ordered. Raising the temperature then results
in an alteration of state (to the liquid-crystalline array) where the
acy} chains are disordered. Much attention has been focused on these
transitions in phase state, most often characterized using techniques
such as differential scanning calorimetry (Figure 53. Abrupt changes in
molecular order of the acyl groups with increasing temperature are
observed for pure components, but addition of other lipids leg.,
cholesterol) eliminate these sharp transitions. The net effect for
actual cellular membranes is that no such sharp transition occurs, and
crystalline domains are unlikely to exist.
Figure 5, Relationship between lipid composition and phase transition.
Careful heating of lipid preparations during analysis by differential
scanning calorimetry tests for the temperature of conversion from the
crystalline to the liquid-crystalline state. Pure lipids (DPPC,
diphosphatidylphosphatidy~choline) undergo this transition at: an unique
temperature, but introduction of increasing amounts of a second 1 ipid
(cholesterol ~ results in a much broader range in the transition
temperature. Adapted from a presentation by Houslay and Stanley (19821.
{~°-RRR??R
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- 161
OCR for page 162
To recapitulate, detailed biophysical studies have established that
quite di';~;iailar lipids, when place in water and allowed to assemble
into aggregate';, assume one of a limited Amber of overall
orientations. Since the hexagonal phase would not provide the essential
permeability barrier for demarcating the cell boundary, this; form cannot
Provide the Thor form in the cell membrane.
The bilayer form is
_
preferred. Recent studies, however, are consistent with the conclusion
that localized and transitory formation of hexagonal forms may be
important in the function of the intact membrane.
These studies of model systems have uncovered another feature of
lipids that may further simplify analysis of the interaction of lipids
in the membranes. This is the concept that lipid interactions, and
therefore patterns of assembly into large scale structures, can be
interpreted in terms of the geometric shape of individual lipid
molecules. -
The primary presentations (Israelachvili, et al., 1980,
Ruypers, et al., 1984 and Gruner, 1985) establish that the dominant
features are the molecular volume, head group area, and depth of the
hydrocarbon side chains. From these considerations, the lipids can be
grouped into molecular shapes and ultimately to predict the Phase into
which they will assemble (Figure 63.
_
A allayer could be envisioned as a
collection of snapes, some with a predisposition to assume one of the
other phase forms (Figure 7~.
Figure 6. Types of lipid packing shapes assumed by various lipids.
Lipids can be grouped according to molecular parameters of molecular
volume, surface area and depth of penetration into the bilayer.
These considerations yield a limiting number of geometric shapes
(cylinders, cones and inverted cones ~ which, in turn, can be related
to the type of polymorphic phase form the system will assume when
placed into water. See text for further details and references.
POLYMORPHIC GEOMETRIC
LIPID PHASE F - M SHAPE
TtOYLC~L 1 - _
TIOYLSE ltlNE ~~, ) ~ ,,, _
IDYLB405'TOL \) i ~ I, l; l,' ~ ~ ' ~ ~ ~
TID - GLYCEROL OF . ....
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~IC£LLaR I - ERTE0 CONE
- 162
l
-
OCR for page 163
Eigure 7. Schematic representation of the assembly of lipid into the
bilayer. A collection of lipid shapes, selected from those presented in
Figure 6, have been assembled to form a bilayer. Such a description
using rigid geometrical forms i'; imperfect in that gaps between lipids
would exist {see outer leaflet). These gaps could be accomodated by the
flexibility of the lipids as well as by the insertion of proteins into
the membrane.
OUTER LEAFLET
INNER LEAFLET
MODEL MEMBRANE (WITHOUT PROTEIN ~
Results of recent studies on differentiation of stem cells to
erythrocytes (Rayler et al., 1985) reveal changes in the "inner vs
outer" display of the polar headgroups of phospholipids (Figure 8~.
Such changes provide one mechanism to greatly alter the surface during
cellular differentiation. A second striking example (Kuypers, et al.
1984; Op den Kamp et al., 1985) is the x~ vitro incubation of
erythrocytes, under conditions where surface lipids were exchanged
without,tranabilayer movement from inner to outer bilayer leaflet, and a
resulting gross alteration of the shape of the erythrocyte. This
alteration can be explained by exchanging 1 ipid molecules residing in
the outer leaflet of the bilayer. Such a 'substitution would result in
an alteration of the surface area of that leaflet, and force a change in
the overall shape of the cell (Figure 9~.
Figure 8. Schematic representation of the changes in the orientation of
E~osphol~ ids during mouse erythroid differentiation. Differentiation
of stem cells into erythrocytes has been intensively studied, with
Friend cells considered by some investigators as a model for an
intermediate stage in the process. Data provided by Rayler, et al.
(1985) establish that the cell types differ in their orientation of
phospholipids toward the outer and inner leaflets.
-
— 163 —
OCR for page 164
~ so -
lo
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lo
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Z 25
ALSO
C
f-RYTHROID DIFFERENTIATION (MOLISE)
STEM CELL ~ | VIRUS
~ tFRIEND CELLS1
BURSt FORMING COLONY fORMING
TOTAL ~OUTSIDE. INSIDE
PC+SM P£ PSond Pl
n ~
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.4
50 OUTER LEAFLET
25
O
25
50 INNER LEAFLET
,.,.
~% .
(ERYTHRocyT£s I
Figure 9. Effect of substitution of lipids of the outer leaflet of
erythrocytes on the overall shape of the cell. Incubation of
erythrocytes with donor vesicles of containing unique
phosphatidylcholine molecules (differing in the acyl substituents, 16:0
vs 18:2) and phospholipid exchange proteins allows selective alteration
of the outer surface of the bilayer (provided the time of the experiment
is limited to less that that required for transhilayer movement). Such
treatment converts the normal erythrocyte (center, bottom) to distinct
forms. Taken from Op den Kamp, et al. (198S) with permission of the
publisher.
OUr5IDE .
not_
6:0,18:1 PC MY t6:0,t8:2;PC
Q In '-~" Qua Q
dlC te:0 PC | = t) C) = ~dICl8:2 PC
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OCR for page 165
Controlled fusion of membranes represents a common membrane event,
often associated with fundamental processes critical for reproduction.
Careful studies of model systems are providing suggestions for the
mechanisms) by which these processes occur; no single mechanism has yet
emerged fro. these studies (see Ohki et al., 1987~. One potential
mechanism involves inverted micelle forms, transiently produced by
relocation of selected lipid components within the bilayer, to provide
those essential features (localized points of stress, integration and
relief of stress) necessary for formation of the new membrane forms
(Figure 10~.
Figure lo. Model fox the role of minor lipids in the fusion of lipid
vesicles. Lipid vesicles (panel a) can come together (panel b) and
bring their lipid bilayers into molecular contact. Minor lipids , with a
propensity for formation of hex II structures, could play a key role in
the interaction of the membrane bilayers (panel c) before separation or
fusion (panel d). Taken with Cullis and Hope (1988) with permission of
the pub] isher.
.~.~ttI~
(S~
Existing data clearly establish that phospholipid asymmetry exists
on cellular membranes and that this condition can persist over the
lifetime of the cell. Such an unequal distribution carries with it the
implication of energy expenditure to maintain disequilibrium. This
could be accomplished by processes such as direct enzymatic action
(phospholipid exchange enzymes moving lipids preferentially to one side
of the bilayer) or synthesis and retention of proteins possessing unique
binding specificities for the phospholipid head groups. Data support
both concepts, but no evidence uniquely supports any single mechanism.
To summarize this section, asymmetric orientation of phoppholipids
on the separate bilayer leaflets exists. Their tendencies to form
unique molecular aggregates can result in atlered cell functions and
shape. These distributions phospholipids provides an excellent
framework for the design and interpretation of experiments dealing with
a variety of membrane related events.
- 16S -
OCR for page 166
LIPID DOMAINS
This concept, also anticipated by Singer and Nicholson, focuses on
the extent to which the membrane is considered to be ~fluid". Recent
data clearly establish that the term must be used carefully. Fluidity
is the inverse of viscosity. The implicit assumption, derived from the
dictionary definition, is that the membrane would have an equivalent
bulk viscosity in all dimensions. Limitations to use of the term for
membrane structures have been discussed (see Lands, 19803. The
predominant restriction is that freedom of motion in the membrane is
very d~.ension-dependent in that lateral (two-dimensional on the
surface), translational (across the bilayer) and rotational (around the
axis of the acyl groups) motions each have their own unique features.
This results in situations where molecules (eg., phospholipids of the
bilayer boundary) can have great freedom of lateral motion but highly
restricted translational motion. The general texts cited in the
preceding section, as well a" the review by Wolf (1988) and sections of
the book by Loew (1988), provide an excellent introduction to the topic.
Even after acceptance of these 1 imitations , it now is apparent that
more stringent restrictions to movement of individual components within
the membrane exist. They include restrictions due to phase segregation,
lipid-lipid interactions, lipid-protein interactions, or morphologically
recognized demarcation between portions of the cell.
Examples of phase separations are found in the data gathered from
model systems of two and three lipid component mixtures. The
predictions from the Gibbs' phase rule establish that at a given
temperature a mixture of components will segregate to form unique gel
phases (each enriched in specific components of the original mixture).
These could tend to form domains within the membrane. The second form,
lipid-lipid interactions, was mentioned previously; an example would be
the apparent preferential interaction of cholesterol with selected
phospholipids that occur as part of the total lipid mixture. Specif ic
lipid-protein interactions also occur, although direct experimental
proof of the extent of the interactions and the factors responsible for
the interaction is lacking. The last feature is recognition of
morphologically distinct portions of the membrane, where points of
restriction (eg., tight junctions) preclude movement from one section of
the surface to the other.
Direct proof of these aspects, much less the molecular basis for
the restrictions, has been difficult to accumulate. However, the
current implication that a variety of important cellular processes
(membrane bound enzyme activation, sperm capacitation, receptor
movements) are regulated in part by these membrane features assures
that the topic will be intensively studied in the near future.-
RELATIVE SPEED OF MEMBRANE EVENTS
Progress in the study membrane events related to reproductive
processes undoubtably will proceed as a form of derivative science,
where data from other areas (eg., erythrocyte and lymphocyte membrane
- 166 -
OCR for page 167
structure-function; model liposome characterization) will ';erve as
examples for experimental design. This is desirable. Progress with
blood cells is easier because these cells have simplified membrane
features and they are actively studied by a relative abundance of
experienced investigators. The next two sections outline precautionary
atatoments regarding this approach.
First, what is the most appropriate display of data to establish
cause-and-effect relationships (Figure 113? The tendency is to array
data in terms of the maximum total response, leading to the assignment
of order of events of A before B before C (in the left panel of Figure
113. This conclusion implies a known stoichiometry of interaction
between the successive elements in the series and that no "overshoot" in
the production of enzyme products occurs. While this may be true for
soluble enzymes, the factors that regulate the activity of
membrane-bound enzymes are less well known. Indeed, since they operate
in a mileau where conformational adj ustment~ may be slowed by
'surrounding molecules, it in highly probable that production of product
beyond that needed for initiation of the next step wil 1 occur . The
essential relationship is to supply ~enough" product to satisfy the
binding needs for the next step in the pathway. One example involves
the sequence of events that follow activation of adenylcyclase, where
absolute amount of cAMP produced in the first step is not important once
~ concentration sufficient to saturate the binding proteins involved in
the next step of the sequence is achieved. The power of retrospective
analysis, possible after detailed study of the sequence, reveals the
quantitative coupling between individual steps. This provides the
revised interpretation of data in the right panel of Figure 11. Thus,
hypotheses should not prematurely die because of our lack of
understanding of the stoichiometry of membrane related events.
The next point of consideration is the effect of the time axis on
analysis of data (Figure 11~. Testing of a hypothesis requires that
experimental observations be made with sufficient rapidity to clearly
establish the kinetic relationships between factors of interest. The
elegant hypotheses related to events of reproduction often are tested by
evaluating features related to changes in membrane permeability,
movement of molecules in/on the membrane or modification of surfaces. A
listing of such events (Figure 12), when scaled to a common axis
(logarithmic, in seconds), is embarassingly informative. It alerts us
to the extreme rapidity of membrane events described in other portions
of this paper and to the fact that all too often the true ';cale of
measurements is inappropriate for the events to be studied.
167 -
OCR for page 168
Figure 11. Effect of selection of axis dimensions on the
_assignment
of sequence in a metabolic process. For ~ simulated system, data for
response of three enzymes (evaluated by assay of products of each) after
addition of a stimulant are given (left panel). For each enzyme, the
cats were normalized to the maximu" Amount of product detected over the
total time of the experiment. Precursor-product assumptions would
suggest the relationship of A before B before C. It is possible that a
detailed examination of the stoichiometry of the individual steps
relative to overall pathway needs (see text for examples) would reveal
that C is produced in vast excess, and A in moderate excess relative to
B. Such considerations would be consistent with the sequence C before A
before 8.
100
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- 158
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TIME 0F INTEREST
OCR for page 169
n9,~7 ~.,~
the time ~c.te-D! evened ~cc~rin~ on
membranes to those of reproduction. Events intern membranes differ
greatly in time, ranging from picoseconds (10 ~ to months (10
seconds) while molecular and organismal events of reproduction are
confined to a much smaller range of time. The effects of such
considerations on the design of experiments are discussed in the text.
Adapted from a presentation by Jain (1987~.
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The comparison in Figure 11 will be used for two illustrations. In
the first, very unique experimental designs are essential for any direct
test of a postulated relationship of regulation of a cellular event
involved in reproduction via a mechanism such as alteration of Nat
- 169
OCR for page 170
movement through channels . Since these processes dif fer in speed by at
least four orders of magnitude, no single incubation will test the
desired relationship. Since direct test is impossible, elimination of
alternative possibilities is most difficult. The required experimental
design, use of several experiments featuring overlapping analyses of
several cellular events to provide the desired temporal relationship, is
rarely used. The second illustrates an opposite effect. Review of many
discussions of tranabilayer movement of phospholipids leaves the
impression that these processes are so slow that they may be of minimal
physiological significance. This may be untrue for events such as the
epididymal maturation of sperm, in that the 7-14 days required for the
process are sufficient to allow for extensive movement of phospholipids
between the inner and outer leaflets of the bilayer.
It is evident that study of membrane events is very immature,
especially for complex membrane processes related to reproduction.
Great care must be taken when adapting hypotheses derived from studies
on more simple systems to assure that the extrapolation is consistent
with additional information about the biological process.
SPATIAL RELATIONSHI PS ON THE MEMBRANE SURFACE
Cell-to-cell interactions, such as sperm-egg binding and fusion,
reflect the end point of an extensive differentiation process whereby
the surfaces are modified to allow specific molecular interactions.
Katz, Cone and colleagues (Katz , et al ., 1987 ; 1988 ~ have calculated the
forces necessary to tether a motile sperm to the oocyte, and provide an
order-of-magnitude estimate of a requirement of up to 10 noncovalent
bonds between the respective surfaces. This serves as a minimum
estimate, since mouse sperm binding to the zone pellucida may ultimately
involve 10-50 x 10p molecules per sperm head (see review by Wassarman,
1987~. Since it is possible to induce specific membrane vesiculation
processes, related to the acrosome reaction, using liposome surfaces
that are much smaller than those of the egg (Graham et al, 1986; Graham
and Hammerstedt, 1988), the following questions merit consideration.
What are the spatial requirements for a specific interaction, with
special emphasis on relating the body of microscopic data with the
emerging biochemical description of the cell surface? Given the large
difference in size of a liposome and an oocyte, are comparisons of
events occurring on each surface directly comparable?
The scale drawings in Figure 13 illustrate both comparisons. On
the left is a model of bovine spend binding to an oocyte, while in the
center (at 10X increase in scale) is binding of a liposome to a bovine
sperm. At this level, direct comparison of events between the sperm
surface and an oocyte vs a 1 iposome appear inappropriate . However, when
viewed from the perspective of the bonds to be formed (right panel, at a
further 100X increase in scale) it is clear that significant
interactions of the sperm surface can occur with either surface. The 10
bonds needed for tethering wil 1 come from portions of the surface that
are indistinguishable using microscopic techniques. These illustrative
examples serve to show the difficulty of direct comparisons of data
gathered using diverse techniques ranging from microscopy to biochemical
and biophysical analyses.
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OCR for page 171
Figure 13, Size relationships of the cells and structures involved in
~ _
sperm egg fusion. In panel A a bovine sperm is shown superimposed on a
bovine egg, in panel B a liposome is shown bound to the sperm head and
in panel C the liposome is shown relative to the bilayer of sperm
surface (see Figure 21. The marker bars illustrate the lOOOX increase
in magnification. The effects of these considerations on the design and
interpretation of experiments is presented in the text.
A
~ ~ BO,OOOnm ;
\
.
B',
(~?11~ (a
2,000nm
~ 1
SUMMARY
Introduction of the fluid-mosaic model of a membrane in 1972
remains a landmark contribution to cell biology. As to be expected,
subsequent contributions have refined the model by reemphasizing and
clearly depicting the modes of molecular heterogeneity possible on any
given surface. These unique orientations and motional properties of the
molecules ultimately will allow discrimination among superficially
similar, but uniquely regulated, membrane events.
Data from model systems aid understanding the potential
characteristics of membrane surfaces involved in fundamental processes
of reproduction. However, the challenge to those interested in
reproduction is to make appropriate extrapolations from a model
systems to the complex cell-cell interactions of interest.
The objective of this overview is to introduce recent contributions
that provide a startpoint for interpretation of data on changes in
membrane composition. As an example, Parks and Ha:mmerstedt ~1985 )
described changes, associated with epididymal transit, in 1 ipid
composition of the plasma membrane overlying the acrosome of ram sperm.
Concomitant with this passage, sperm acquire the capacity to undergo the
acrosome reaction (Williams, et al., 19873. The challenge is to devise
an appropriate means to test for a relationship between changes in
structure (lipid and protein) and changes in function (ability for
specific vesiculation). Unique interpretations have not been provided,
since if the reported lipid changes are considered in terms of the
concepts of membrane fluidity, ~ prediction of an increase, decrease or
no change in this parameter can be made! The conclusion drawn depends
on the model system accepted for comparison. The alteration in 1 ipid
composition should introduce a change in the asymmetry of lipids across
the bilayer. This could have extreme ef feats on the interaction of thi s
- i71 -
OCR for page 172
sperm 'surface with other surfaces. Distinctions among the various
possibilities requires careful extrapolation from model studies, with a
greater emphasis on direct evaluation of the physical features of the
membranes of interest.
ACKNOWLEDGEMENTS
Financial support was provided by the National Institutes of Health
(HD 13099) and the United States Department of Agriculture (GAM
85025353. Dr. James Graham provided essential suggestions for Figures
12 and 13.
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175 -
.
Representative terms from entire chapter:
membrane events