biomedicines

Review
Fifty Years of the Fluid–Mosaic Model of Biomembrane
Structure and Organization and Its Importance in Biomedicine
with Particular Emphasis on Membrane Lipid Replacement
Garth L. Nicolson 1, * and Gonzalo Ferreira de Mattos 2

1 Department of Molecular Pathology, The Institute for Molecular Medicine, Huntington Beach, CA 92647, USA
2 Laboratory of Ion Channels, Biological Membranes and Cell Signaling, Department of Biophysics,
Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay; ferreiragon@gmail.com
* Correspondence: gnicolson@immed.org; Tel.: +1-949-715-5978

Abstract: The Fluid–Mosaic Model has been the accepted general or basic model for biomembrane
structure and organization for the last 50 years. In order to establish a basic model for biomembranes,
some general principles had to be established, such as thermodynamic assumptions, various molecu-
lar interactions, component dynamics, macromolecular organization and other features. Previous
researchers placed most membrane proteins on the exterior and interior surfaces of lipid bilayers to
form trimolecular structures or as lipoprotein units arranged as modular sheets. Such membrane
models were structurally and thermodynamically unsound and did not allow independent lipid and
protein lateral movements. The Fluid–Mosaic Membrane Model was the only model that accounted
for these and other characteristics, such as membrane asymmetry, variable lateral movements of
membrane components, cis- and transmembrane linkages and dynamic associations of membrane
Citation: Nicolson, G.L.; Ferreira de components into multimolecular complexes. The original version of the Fluid–Mosaic Membrane
Mattos, G. Fifty Years of the Model was never proposed as the ultimate molecular description of all biomembranes, but it did
Fluid–Mosaic Model of Biomembrane provide a basic framework for nanometer-scale biomembrane organization and dynamics. Because
Structure and Organization and Its this model was based on available 1960s-era data, it could not explain all of the properties of various
Importance in Biomedicine with biomembranes discovered in subsequent years. However, the fundamental organizational and dy-
Particular Emphasis on Membrane namic aspects of this model remain relevant to this day. After the first generation of this model was
Lipid Replacement. Biomedicines 2022, published, additional data on various structures associated with membranes were included, resulting
10, 1711. https://doi.org/10.3390/
in the addition of membrane-associated cytoskeletal, extracellular matrix and other structures, spe-
biomedicines10071711
cialized lipid–lipid and lipid–protein domains, and other configurations that can affect membrane
Academic Editor: Vijay Kumar dynamics. The presence of such specialized membrane domains has significantly reduced the extent
Thakur of the fluid lipid membrane matrix as first proposed, and biomembranes are now considered to be less
fluid and more mosaic with some fluid areas, rather than a fluid matrix with predominantly mobile
Received: 20 June 2022
Accepted: 10 July 2022
components. However, the fluid–lipid matrix regions remain very important in biomembranes,
Published: 15 July 2022 especially those involved in the binding and release of membrane lipid vesicles and the uptake of
various nutrients. Membrane phospholipids can associate spontaneously to form lipid structures
Publisher’s Note: MDPI stays neutral
and vesicles that can fuse with various cellular membranes to transport lipids and other nutrients
with regard to jurisdictional claims in
into cells and organelles and expel damaged lipids and toxic hydrophobic molecules from cells and
published maps and institutional affil-
tissues. This process and the clinical use of membrane phospholipid supplements has important
iations.
implications for chronic illnesses and the support of healthy mitochondria, plasma membranes and
other cellular membrane structures.

Copyright: © 2022 by the authors. Keywords: lipid interactions; membrane domains; extracellular matrix; lipid rafts; membrane fusion;
Licensee MDPI, Basel, Switzerland. membrane structure; membrane dynamics; cytoskeletal interactions; membrane vesicles; endosomes;
This article is an open access article exosomes; detoxification; chronic medical conditions
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Biomedicines 2022, 10, 1711. https://doi.org/10.3390/biomedicines10071711 https://www.mdpi.com/journal/biomedicines

Biomedicines 2022, 10, 1711 2 of 25

1. Introduction: Barriers, Cellular Compartments and Biomembrane Structure

When exogenous or extracellular molecules, including water, ions, nutrients, sugars,
proteins, glycoproteins, lipids, lipoproteins and other components, such as extracellular
structures, stroma, extracellular matrix, lipid vesicles, viruses, microorganisms, and other
cells approach a cell, they first encounter cell membranes or cell membrane-associated
structures [1,2]. Cell membranes and their associated structures are the most important
barriers to cell entry and exit of molecules, ions, and other structures, allowing a unique
intracellular microenvironment [2–4]. The interactions of extracellular molecules and
stromal structures and cell membranes are important in maintaining the exclusion of
extracellular molecules, and they are also critical in segregating membrane molecules,
regulating cell polarity, modulating the exchange of molecules, initiating cellular signaling,
and moderating the responses to and maintenance of many normal cellular processes [3–5].
Although cell membranes provide cells with barriers and compartmentalization, they
also support cellular and tissue continuity. The barrier functions can also be quite selective
and adjustable, and thus cells are capable of selectively transporting particular nutrients
and other substances into cells and eventually into various cellular organelles, as well
as transmitting certain molecular signals into and out of cells. These molecular signals
and effectors can be various secreted ions and molecules, lipid-associated structures, lipid
vesicles, such as exosomes, and other substances that can find their way to adjacent cells,
tissues and distant organs, and in the process initiate changes in cell and tissue microenvi-
ronments. Inside cells, various intracellular membranes are responsible for the segregation
of enzymatic processes, the biosynthesis and transport of various molecules, and generally
the separation of basic cellular functions, such as energy production, replication, secretion
and other cellular activities [3–6].
Although each biomembrane is unique in its detailed structure, composition, dynamics
and function, there are some general structural and organizational principles that should
apply to all cellular membranes and be present in any biomembrane model.
Biomembrane General Principle No. 1: The importance of noncovalent forces in establishing
and maintaining nanoscale biomembrane structure cannot be underestimated.
Biomembrane General Principle No. 2: Lipid bilayers are essential in providing a basic matrix
and continuity for biomembranes.
The first two general principles necessary for any biomembrane model should cover
the most basic aspects of cellular membranes. Cell membranes are macromolecular struc-
tures that are present in aqueous solutions and whose components are largely held together
by noncovalent forces. At their most basic organizational level they can be thought of as dy-
namic barrier matrix structures made up of amphipathic lipid and protein components that
spontaneously associate into largely noncovalently bound macro-structures that exclude
water interactions on their hydrophobic surfaces. In contrast, the hydrophilic portions of
their structures interact with the aqueous environment and other hydrophilic and ionic
molecules [3,5–8]. This concept was implied by the experiments of Langmuir, who studied
the formation of oil layers on aqueous surfaces [7]. Using this methodological approach, it
was estimated that red blood cells are surrounded by two layers of membrane lipids [9].
This was also consistent with Fricke’s findings from cell membrane capacitance experi-
ments that estimated that cell membranes should be approximately 4 nm thick [10]. The
historical representations that cell membranes are basically composed of a phospholipid
bilayer matrix plus some membrane proteins has been reviewed elsewhere [11]. In one
notion of how cell membranes are organized, it was proposed that cellular membranes
are basically phospholipid bilayers that interact with flattened or beta-sheet-structured
proteins via the hydrophilic head groups of membrane phospholipids and certain amino
acids [12]. Visualization of this structure, primarily by transmission electron microscopy
of erythrocytes and other cells fixed and stained with heavy metals and embedded in
polymeric resins and transversely thin-sectioned, revealed what appeared in cross section
to be tri-molecular layers of membrane components. This visual representation was pro-
Biomedicines 2022, 10, 1711 3 of 25

moted as support for the basic organization of cell membranes as a trimolecular, layered
structure composed of protein–lipid–protein units (the Unit Membrane) [13]. A competing
membrane model was subsequently proposed that was based on a monolayer of repeating
subunits of lipoproteins without a matrix composed of a phospholipid bilayer [14]. None
of the ‘sandwich’ or ‘lipoprotein subunit’ models of cell membrane structure proved to
be correct [8].
Biomembrane General Principle No. 3: Membrane components attempt to maximize compati-
ble molecular interactions in order to approach the lowest free energy state.
Any model for biomembranes should consider the most important forces that hold
membranes together and resist the natural tendency to disorganize over time. The concept
of exploiting noncovalent forces that match the hydrophobic portions of each integral
membrane component and minimize nonmatching interactions drive the spontaneous
assembly of membrane lipids and proteins [15,16]. This implies that biomembranes are
composed of a lipid matrix composed of amphipathic phospholipids that self-assemble to
form a lipid bilayer due to the free energy provided by the hydrophobic effect and van
der Waals forces [15,16]. Into this lipid bilayer matrix, integral membrane proteins are
thought to assemble and interact with membrane lipids mainly via hydrophobic forces and
much less by hydrophilic forces between lipid head groups and the membrane proteins’
hydrophilic amino acids [3,8,15–18]. The physical state of membrane phospholipids is
important in this process because the insertion of integral membrane proteins into a lipid
bilayer matrix may be limited to regions of membrane where the lipid matrix allows
protein penetration and intercalation into the lipid bilayer. In the process, membrane
protein–lipid hydrophobic interactions must be thermodynamically favorable. Thus, in
the membrane regions where phospholipid and protein molecular sorting can occur, the
hydrophobic and van der Waals forces can be maximized, and the lowest free energy
state can be approached [7,8,16]. Thus, the lateral, independent movements of membrane
components are possible in a fluid matrix [3,8,15,16,18]. (The different interactions of lipids
with other lipids to form various domains of differing lipid compositions will be discussed
in a subsequent section.) The most stable membrane structure is one that maximizes
hydrophobic interactions, stabilizes ionic interactions and couples different ionic charges
in an attempt to approach the lowest free energy state [8,16].
Biomembrane General Principle No. 4: Membrane proteins are a large and diverse group of
molecules that can be placed into different classes depending on their interactions with membrane
lipids and their abilities to intercalate into the membrane lipid bilayer matrix.
In this general principle of biomembrane structure, we consider the protein compo-
nents and how they are incorporated into the overall structure. There are large numbers
of unique proteins that are present in various cellular membranes [15,19]. These mem-
brane proteins can be assigned to different categories or classes based on their amino
acid sequences, functions and properties. Operationally, in the Fluid–Mosaic Membrane
model, they were consigned to three simple classes: integral, peripheral [8,15], and (added
later) membrane-associated proteins [17,18]. The classic integral (or intrinsic) membrane
proteins were depicted as globular, amphipathic proteins that were intercalated into lipid
bilayers and stabilized mainly by hydrophobic forces (Figure 1) [8]. In this model, the
integral membrane proteins were thought to penetrate into the membrane lipid bilayer
matrix to various degrees, from completely spanning the membrane to barely infiltrating
into the lipid bilayer [8,15]. These membrane proteins and glycoproteins have extensive
alpha helical regions and lipid-interacting structures at their surfaces, and they represent
families of transport, adhesion, signaling and other molecules that that can be potentially
modulated by their adjoining lipids. At the time, peripheral membrane proteins were
proposed to be attached to membranes mainly by electrostatic or other forces [8]. These
peripheral membrane proteins were purported to be removeable from membranes without
destroying basic membrane structure and continuity [8]. They were subsequently found to
Biomedicines 2022, 10, 1711 4 of 25

serve as important components in providing membrane stability, deformation, curvature,

scaffolding and other characteristics, such as attachment points for enzymes and signaling
complexes [17–19]. A few years after the publication of the Singer–Nicolson model [8], the
other category was added: membrane-associated proteins [17]. Membrane-associated pro-
teins can be globular in structure, but generally they are not amphipathic and not associated
with the hydrophobic membrane lipid matrix, nor are they thought to be bound by mainly
electrostatic forces. These proteins can also be transiently associated with membranes
via interactions with integral membrane proteins or linked to lipid molecules instead of
being intercalated to various degrees into the membrane lipid matrix and stabilized by
hydrophobic forces [17,18]. The membrane-associated proteins were alleged to dynamically
and intermittently provide connections between cell membranes and other intracellular
components at the inner membrane surface and extracellular and stromal components at
the outer membrane surface. Examples of membrane-associated components at the inner
membrane surface could be intracellular proteins, enzymes, protein complexes and cy-
toskeletal elements or, at the outer surface, extracellular stromal and matrix elements [17,18].
Membrane-associated proteins are now thought to assist in maintaining membrane shape,
structural integrity and dynamics as well as to provide linkages to other intracellular and
extracellular proteins and glycoproteins [3,5,17–19].

Figure 1. The Singer–Nicolson Fluid–Mosaic Membrane Model of cell membrane structure as

proposed in 1972. In this static view of a fluid cell membrane, the solid bodies with stippled cut
surfaces represent globular integral membrane proteins randomly distributed in the plane of the
membrane. Some integral membrane proteins form specific integral protein complexes, as shown in
the figure. Integral proteins are represented as intercalated into a fluid lipid bilayer, but peripheral
membrane proteins are mentioned but not shown, nor are tightly-bound lipids. Further, the figure
does not contain other membrane-associated structures or membrane domains (Redrawn from Singer
and Nicolson [8]).

Biomembranes in general are dynamic structures that can be disturbed, distorted,

deformed, compressed or expanded by different forces [3,16–18]. Indeed, certain periph-
eral membrane proteins can bind to and cause membrane deformability by binding to
biomembranes, in the process causing membrane curvature as a result of flexing and
bending of membranes to fit the structures of these peripheral proteins [3,16–18,20,21].
In contrast, membrane-associated proteins are thought to act indirectly on membranes,
usually through intermediate protein or lipid attachments. Membrane peripheral and
membrane-associated proteins should be removable from membranes without disrup-
Biomedicines 2022, 10, 1711 5 of 25

tion of the membrane’s basic structural integrity and continuity of its hydrophobic ma-
trix [3,8,17,18]. Membrane-associated proteins can be present in the cell cytoplasm or
outside cells and include cytoskeletal and signaling structures bound at the inner cell
membrane surface or extracellular matrix and stromal components interacting at the outer
extracellular membrane surface. These membrane-associated components can be quite
dynamic and can stabilize or destabilize cellular membranes and their connections to other
intracellular or extracellular structures [3,17,18]. Alternatively, they can be involved in
stabilizing the dynamic properties of membranes and preventing membrane components
from undergoing various lateral movements and consigning them to certain spaces, or they
can participate in the directional movements or translocation of membrane complexes via
energy-dependent processes [3,17,18]. Membrane-associated proteins are involved in main-
taining or eliciting certain specific cellular processes, including: cell adhesion, stabilization,
motility, growth, endocytosis, exocytosis and other important cellular functions [3,5,16–18].

2. The Fluid–Mosaic Model of Biomembrane Structure

Although various models of biomembrane structure have been presented in the
literature over the last 50-years, the most accepted nanometer scale model of basic cell
membrane structure remains the Fluid–Mosaic Membrane Model (Figure 1) [8]. This model
has been criticized as an oversimplified and obsolete scheme for explaining the complex
nature of cellular membranes and their hierarchical structural organization, as well as for
its failure to account for some of the dynamic properties and domain organizations found
in certain biomembranes [22–24]. In its defense, however, the Fluid–Mosaic Membrane
Model was never intended to explain all aspects of membrane structure and dynamics,
especially those discovered after 1972. Instead, it was generated to provide a basic minimal
framework of cellular membrane organization and dynamics, not as an ultimate future
description for all of the potential molecular arrangements and subtleties present in various
cellular membranes.
Biomembrane General Principle No. 5: When proposing a model for basic biomembrane struc-
ture and organization, it should be consistent with current data as well as with future discoveries.
Biomembrane General Principle No. 6: Biomembranes are asymmetric in their distribution of
membrane proteins and glycoproteins, certain lipids and glycolipids and peripheral and membrane-
associated proteins on inner and outer membrane surfaces.
These two general principles incorporate some basic elements of biomembranes that
are based on the published evidence that accumulated in the years before 1972. The Fluid–
Mosaic Membrane Model was put forward to provide a simple framework for the basic
organization of biomembranes, not as a detailed explanation of the structure, asymmetry,
dynamics and functions of every biomembrane [8]. We believe that it has accomplished
this goal for the last 50 years, but it obviously has required periodic updates [3,17,18]. The
Fluid–Mosaic Membrane Model has demonstrated its usefulness, but only as a simplified,
nanoscale representation of rudimentary biomembrane structure. In that respect, it still
represents some of the more important elements of cell membrane architecture, including
continuity, cooperativity and asymmetry of biomembranes, as well as some aspects of
membrane dynamics [3,17,18,25].
The original Fluid–Mosaic Model accounted for membrane asymmetry by estimating
the enormous amount of free energy required to flip amphipathic membrane compo-
nents from one side of a lipid bilayer to the other side [8]. Every cell membrane studied
thus far has been found to be asymmetric in terms of the display of membrane compo-
nents on the interior and exterior sides of membranes, especially those that have attached
carbohydrates [3,8,11,15–18,21,26]. This characteristic of biomembranes appears to be uni-
versal [18,26]. It makes perfect sense to have asymmetric structures that separate different
cell compartments.
Biomedicines 2022, 10, 1711 6 of 25

Biomembrane General Principle No. 7: There is no universal membrane model that can explain
or predict every newly discovered aspect of biomembrane structure, function or dynamics.
We admit that it is virtually impossible to incorporate all of the data published over
the years on biomembranes into a universal model of biomembrane structure. The goal
here is to come up with a reasonable solution that fits best with the available data. With the
limited data available 50 years ago, only a few of the potentially vast number of biomem-
brane characteristics could be discussed at the time in any detail [8]. Some membrane
elements were briefly mentioned but not presented graphically in the original schematic of
a biomembrane (Figure 1), such as membrane asymmetry, specialized lipid environments
surrounding membrane proteins, and other characteristics [8]. Unfortunately, this has led
to some quite literal interpretations of the Fluid–Mosaic Membrane Model and, we feel,
undeserved criticism [22,23].
Although the essential elements of the Fluid–Mosaic Membrane Model have proven
to be remarkably consistent with experimental findings at the nanoscale level over the
last 50 years, it was inevitable that the original model could not explain all of the newly
discovered properties of membranes, including recent findings on the fine structure and
dynamics of protein and lipid components [3,18,22–31]. Importantly, the concept that
membrane mosaic structures and membrane domains, such as lipid rafts and membrane
protein complexes as well as cell membrane-associated structures, such as cytoskeletal
elements and other structures, were essential in controlling membrane properties and
directing the dynamics of certain cell membrane components. These were not features
found in the original model, and many of these new findings were made decades after
the publication of the original Fluid–Mosaic Model [3,18,22–31]. This has resulted in the
suggestion that several membrane models are necessary to explain basic biomembrane
structure and dynamics [22], or that there are no general membrane models that can
adequately describe the structure and dynamics of biomembranes [23]. We understand
the need to constantly update existing proposals. Moreover, we recognize the difficulty
in presenting an accurate model for biomembrane structure and dynamics that takes into
account all of the data accumulated since 1972 [3,18].
Biomembrane General Principle No. 8: Biomembranes appear to be much more complex,
compact and more mosaic than presented in the original Fluid–Mosaic Membrane Model.
There is a misconception over the first presentation of the Fluid–Mosaic Membrane
Model (Figure 3 of Ref. [8], redrawn as Figure 1 in this contribution) that has persisted
over the years since its publication in 1972. That is, the densities of protein components
in biomembrane models have increased with time. Over the last 50 years. various up-
dates of the Fluid–Mosaic Membrane Model have gradually refined the original model of
biomembrane structure and dynamics into far more complex, much less homogeneous,
and more densely packed (more mosaic) models, as emphasized by the authors [3,18] and
Goñi [28]. Compared to the original biomembrane scheme, shown here as Figure 1 [8],
newer membrane models depict biomembranes as more mosaic in nature with many fewer
areas of fluid membrane lipid regions [3,18,22–30]. All of the newer proposals on biomem-
brane organization also contain additional information not shown in the original model
(Figure 1), such as representations of lipid–lipid, protein–protein and lipid–protein associa-
tions into membrane domains of various sizes and surrounded by specific combinations
of lipids as well as nano- and micro-sized complexes within specialized domains, and
their segregation and regulation by transmembrane forces. Importantly, all of the newer
biomembrane models now include membrane-associated structures on the cytoplasmic
side and in the extracellular environment that are capable of immobilizing or alternatively
mobilizing large portions of membrane. Most mammalian cells are located in tissues
where polarity and cellular and extracellular and stromal interactions are important in
segregating and maintaining tissue organization and cellular networks. In addition, newer
information on transmembrane signaling complexes, membrane component interactions
and dynamic changes in membrane organization, along with other additions had to be
Biomedicines 2022, 10, 1711 7 of 25

accommodated [18–32]. These additions over the years have made biomembrane orga-
nizational schemes much more complex and compact (more mosaic) than the original
Fluid–Mosaic Model (for example, Figure 2).

Figure 2. An updated, static representation of the Fluid–Mosaic Membrane Model with more
detail than presented in the original 1972 Singer and Nicolson model [8]. The cell membrane of a
generic tissue cell is depicted with various lipid and protein domain structures as well as membrane-
associated cytoskeletal and extracellular structures. The cell membrane has been peeled back at the
left in order to reveal underneath the plasma membrane. Membrane-associated cytoskeletal elements
can be arranged to form potential barriers (‘corrals’) that could possibly limit the lateral mobilities
of some of the integral transmembrane proteins. In addition, membrane-associated cytoskeletal
structures can indirectly interact with some of the integral membrane proteins at the inner membrane
surface along with stromal or extracellular matrix components at the outer surface. Although this
static diagram presents some of the possible mechanisms of integral membrane protein mobility
restraint, it does not accurately represent the dynamic changes in membrane components, the sizes
and structures of phospholipids and lipid domains, integral and peripheral membrane proteins or
membrane-associated cytoskeletal structures. It also does not reflect the actual crowding or high
density of membrane components. (Modified from Nicolson [18]).

During the last decade, it has become fashionable to position most biomembrane
lipids and proteins into less freely-mobile domains, such as lipid-rafts and lipid–protein
complexes, or specialized membrane domains linked to cytoskeletal elements. Hence the
mosaic nature of cellular membranes has now been accentuated over the fluid nature of
membranes [3,18,28]. Although newer biomembrane models contain fewer fluid areas of
freely mobile membrane lipids and proteins than presented in the original Singer–Nicolson
model [3,18,22,24,28], the basic nanoscale organization first presented in the Fluid–Mosaic
Membrane Model as diverse amphipathic proteins intercalated to various degrees into a
lipid bilayer matrix has generally survived [3,18,22–30].
Biomembrane General Principle No. 9: Biomembranes appear to have a more complex multi-
component hierarchical organization than originally envisioned
This general principle of biomembranes signifies that for over 50 years new data
on biomembrane structure and organization have necessitated some adjustments in the
original Fluid–Mosaic Model. As described above, current biomembrane models are more
Biomedicines 2022, 10, 1711 8 of 25

crowded and complex (more mosaic) than presented in the original Singer–Nicolson pro-
posal [3,18,28,29]. To add to this complexity, Kusumi and his colleagues have advanced
the concept of a dynamic hierarchical cell membrane structural organization [24,25]. This
has made a complicated description of cell membrane organization even more layered
and complex in order to incorporate recent data on the presence of various macromolec-
ular structures (superstructures) in some biomembranes. The various macromolecular
structures appear to place restrictions on the distribution and mobility of some mem-
brane components [24,25]. This will be described in more detail in a subsequent section of
this review.
Any reasonable schematic of biomembrane organization should depict the nonran-
dom sorting and the various mobilities and distributions of different membrane compo-
nents [27,29,31]. The spontaneous, dynamic sorting of membrane components into various
membrane domains was thought to be based, at least initially, on hydrophobic and some hy-
drophilic interactions [3,15,18,32]. Such dynamic sorting avoids hydrophobic mismatches
between various lipids and lipids and proteins, thus preventing unsustainable membrane
distortions or areas of membrane weakness [32].
In the original Fluid–Mosaic Model, the presence of some oligomeric protein/glycopr-
otein structures in the membrane was first proposed (see Figure 1) [8]. Some early evi-
dence (discussed in [8]) was the discovery of different cell surface antigen distributions—
dispersed [33] or micro-clustered [34]—on the same cell type. That notion has now
become more refined based on evidence gathered with new technologies developed to
study the localization and dynamics of single molecules on cell surfaces at the nanometer
scale [29,31,35]. For example, Garcia-Parajo and colleagues found that many, if not most,
cell membrane proteins and glycoproteins exist in small mobile nanostructures or nanoclus-
ters in the membrane [29]. Using Förester Resonance Energy Transfer (FRET) combined
with single particle tracking and fluorescence microscopy, Ma et al. studied the associations
of neighboring membrane proteins and their clustering events at high spatial and temporal
resolutions [35]. By plotting the individual mobilities and clustering events on live cells,
Pageon et al. found that certain receptors were already present in ‘nanoclusters’, and these
dense receptor clusters were important in providing the greatest signaling efficiencies [36].
Membrane domain dynamics involve lipid–lipid and lipid–protein interactions as well as
inner membrane surface protein scaffolding and the involvement of membrane-associated
elements [37]. This will be discussed again in a following section.
Over time, the basic nanoscale organization of cell membrane models has evolved
significantly from the original models of rather homogeneous-looking structures, such as the
diagram shown in Figure 1 [8], into more heterogeneous models that are still dynamic yet
contain mosaic structures that comprise specific domains of varying sizes, compositions and
component mobilities. Some of these structures can form into specific membrane regulatory
and mechanical platforms that are linked to various intra- and extracellular components
(cytoskeleton structures, stromal components, extracellular matrix, etc.) [3,18,22,24,25].

3. Some Important Interactions of Proteins and Lipids in Biomembranes

In the original Fluid–Mosaic Membrane Model, membrane components were, in
general, portrayed as primarily randomly distributed and unrestrained in their lateral
movements [8]. However, as mentioned in Section 2, certain properties, such as the variable
lateral mobilities of many membrane components, are now assumed to be part of the
model [3,17,18]. Within a few years after the original model was presented, these concepts
were incorporated into an updated Fluid–Mosaic Membrane Model [17].
Biomembrane General Principle No. 10: Changes in the compositions of certain asymmetri-
cally distributed membrane lipids can modify the physical characteristics of biomembranes.
This general principle of biomembranes reflects the many studies on the properties of
membrane phospholipid and other membrane lipids over the last 60 years. Biomembranes
are known to contain hundreds of different types of lipids, most in minute concentra-
Biomedicines 2022, 10, 1711 9 of 25

tions [11]. We do not know the functional consequences of having very-low-abundance

membrane lipids in cellular membranes, although the complete absence of specific lipids
has known clinical consequences. Moreover, it is accepted that certain specific membrane
phospholipids can form specialized zones around the hydrophobic surfaces of membrane
proteins, possibly due to their stronger interactions with the hydrophobic surfaces of
membrane integral proteins and glycoproteins, and to a lesser degree to hydrophilic inter-
actions [32,38–40]. In addition, certain membrane phospholipids have been found to be
asymmetrically present on the inner and outer leaflets of plasma membranes and also un-
evenly distributed in the membrane plane in certain lipid domains, and this could have
important consequences on membrane curvature, flexibility and other properties [3,32,38–41].
In addition to membrane phospholipids, other lipids are also distributed nonrandomly in
various cellular membranes. For example, cholesterol is intercalated into biomembrane
bilayers and can modify the characteristics of membranes and change lateral lipid distri-
butions and dynamics [32,38,39]. Cholesterol is often found to be enriched within specific
membrane domains [38,41]. Its distribution is thought to be due, in part, to its affinity for
both the fluid and solid phases of membrane phospholipids [38,41]. Cholesterol partitions
into liquid-ordered and disordered lipid phases to roughly the same extent, but this parti-
tioning can modify the properties of dissimilar membrane lipid phases [38,41]. Changes in
the compositions of membrane phospholipids can also modulate certain physical proper-
ties of membranes [42]. For example, changing cholesterol content can modify membrane
lateral elasticity, whereas ceramides and lysophospholipids are known to induce changes
in membrane curvature [38,41,42].
Biomembrane General Principle No. 11: Biomembrane lipids in specialized lipid–protein
domains are essential in maintaining membrane structure and function.
Specific membrane lipids, for example sphingolipids, are important in the formation
of ordered membrane lipid mosaic domains or ‘lipid rafts’ [38,43–47]. With phosphatidyl-
choline, sphingomyelins constitute more than one-half of the cell membrane phospho-
lipids and are the most important companions of cholesterol in lipid domains or lipid
rafts [47,48]. Small, ordered membrane rafts/domains assemble by preferential associa-
tions of cholesterol and saturated lipids. These rafts/domains are generally surrounded by
liquid-phase lipids, and thus they are able to undergo membrane lateral movements [46,47].
Lipid rafts/domains can also selectively recruit additional lipids and proteins into their
structures [30,43–47]. Not all of the lipids within such mosaic domains are completely
immobilized—they are still rotationally and laterally mobile to some degree and capable
of slowly exchanging their lipids with bulk membrane lipids as well as with lipids in
other membrane domains [30,46,47]. The overall sizes of lipid domains, such as lipid rafts,
are usually less than 300 nm in diameter; most are within 10–200 nm in diameter, with
some slightly larger [49–51]. However, they can undergo domain clustering induced by
protein–protein and protein–lipid interactions, and the result is an increase in domain
diameter to approximately micrometer size (>300 nm) [30,50].
Lipid rafts/domains usually contain some peripheral membrane proteins and lipid-
linked proteins as well as some integral membrane proteins, and these mixed lipid–protein
domains are not static [50,51]. Lipid rafts/domains undergo changes in lipid and/or
protein compositions over time and can convert these membrane platforms into functional
signal transduction domains. Eventually, the transmembrane-coupled rafts/domains can
initiate various functions, such as immune receptor signaling, host–pathogen reactions,
cell-death regulation and other cellular processes [43,45,46,50,51].
Membrane proteins can have profound effects on biomembranes and on lipids within
lipid rafts/domains. They can deform membranes and cause reorganization of membrane
lipids to form new membrane domains as well as regulate various membrane properties,
such as charge density and diffusion rates [21,52]. When integral membrane proteins
interact with membrane lipids in biomembranes, portions of their structures must directly
interact with the acyl chains of membrane phospholipids or other hydrophobic regions of
Biomedicines 2022, 10, 1711 10 of 25

other molecules. This is accomplished by hydrophobic matching between the hydrophobic

regions of proteins and lipids [32,38]. Hydrophobic matching between the hydrophobic core
of the lipid bilayer and hydrophobic stretches of amino acids in integral membrane proteins
results in stable hydrophobic interactions by the exclusion of water. If the hydrophobic
portions of their structures are mismatched, elastic distortion of the lipid matrix around
the integral membrane protein occurs [32,38]. This can induce protein conformational
changes that can affect protein function and protein–protein and protein–lipid interactions.
Membrane proteins can also aggregate to form super-domains in membranes. In addition,
there are other physical forces, such as lateral pressure forces, lateral phase changes,
membrane curvature, ionic interactions and other forces, that are important in regulating
membrane structure, function and dynamics [52–54].

4. Membrane-Associated Cytoskeletal and Extracellular Matrix Interactions with

Biomembranes
Negligible information was available on membrane interactions with intracellular
cytoskeleton networks and extracellular matrix elements at the time of the original Singer–
Nicolson publication [8]. Although such interactions were assumed to be important in
the attachment of cells to substratum and stroma, at the time, the components involved
in these interactions were not well-characterized [55]. That cell membrane-associated
interactions could alter cell membrane macrostructure by restricting the dynamics or lateral
movements of membrane proteins and glycoproteins and segregating them into membrane
domains was virtually unknown at the time. In addition, important membrane properties,
such as cytoskeleton–membrane linkages are now known to be involved in immobilizing
membrane domains as well as moving domains with the assistance of energy-dependent
cytoskeletal processes [3,17,18,27–31,56,57]. Indeed, cytoskeletal and extracellular elements
are now known to be essential in maintaining cell polarity and tissue organization [56–64].
Biomembrane General Principle No. 12: Biomembranes are not automatous structures—their
components and domains are linked and integrated with various intracellular and extracellular structures.
We will now consider some of the more complex properties of biomembranes that
are related to their many functions. There are a number of tissue and cellular properties,
such as cell orientation, cell adhesion, cell movement, cell stabilization, cell communication,
cell differentiation and many other properties, that are driven or stabilized by membrane-
associated cytoskeletal interactions with membrane domains [3,17,18,25,51,56–60,64,65].
Cell membrane receptor clustering, domain formation, submembrane plaque assembly,
membrane distortion and internalization and recycling of membrane components are all
important in maintaining normal cellular physiology [3,18,25,30,36,44–46,49,60]. Therefore,
the early addition of membrane-associated cytoskeletal interactions to various versions
of the Fluid–Mosaic Membrane model was considered very important [17,18]. The distri-
butions and mobilities of integral membrane components can be modified or selectively
anchored by cytoplasmic membrane-associated cytoskeletal components or by extracellular
interactions (cell–cell, cell–matrix or cell–stromal interactions), resulting in cell membrane
domain immobilization [56,58–60]. Such interactions also contribute to cell polarity and
tissue organization [3,56,60].
Biomembrane General Principle No. 13: Biomembranes possess specialized domain structures
for extracellular and intracellular signaling and communication.
One of the more important concepts in describing biomembranes over the years has
been describing the linkages between the structural, organizational and dynamic aspects
of membranes and the functional properties of cells, such as the communication of specific
signals. For example, cell signaling and inter-cell communication are essential for maintaining
various tissues and circulating cells. Cellular communications can take many different forms,
for example, ions and transmitters in nerve transmission, hormones and other mediators,
immune communication signals and many other examples, and these often involve special-
ized membrane domains. Often, individual communications and signals are transmitted
Biomedicines 2022, 10, 1711 11 of 25

into the cytoplasmic compartments of cells via dynamically assembled cell surface recep-
tors and membrane-associated complexes in domains that dynamically include cytoskeleton
systems [18,24,25,27,29,44,51,58,59]. As mentioned above, the cytoskeleton can also gener-
ate mechanical forces that can laterally move membrane complexes, membrane platforms,
domains and even entire cells, or can inhibit their movements to help cells resist exterior
mechanical forces [56,58–60,66]. The serial assembly of cytoplasmic proteins and cytoskeletal
elements in and around membranes into specialized domains may be essential in the conver-
sion of biochemical signals into mechanical forces that can influence cellular behavior, such
as cell movements and organization of tissue structure [25,28,56,58–60]. There are a variety
of membrane peripheral proteins and enzymes that have been identified as components
involved in membrane–cytoskeletal interactions [24,25,28,51,56,58–60]. Membrane lipids are
also involved in membrane–cytoskeletal interactions, resulting in the formation of specialized
lipid signaling domains usually known as lipid rafts [30,44–46,49–51]. Although cytoskeletal
involvement in specialized lipid domains was unknown at the time that the original Fluid–
Mosaic Membrane model was proposed, the formation of lipid domains in biomembranes
was foreseen years before actual experimental evidence for their existence was obtained [17].
Lipid membrane domains appear to form spontaneously as dynamic structures that result
from the specific sorting of bulk lipid components, with some specific lipid-binding proteins or
glycoproteins in structures held together mainly by noncovalent bonds [43–47,49–51]. It is not
known if ligand or ion binding plays a role in lipid domain or lipid raft formation, but these
events are likely to occur after these structures have spontaneously formed the membrane.
The involvement of cytoskeletal transmembrane interactions with an assembled lipid raft or
domain is likely a secondary event for initiation of transmembrane signaling [46,50,51]. The
initial part of this process appears to be the presence of glycosylphosphatidylinositol (GPI)
anchors at the cell surface in lipid domains or rafts [46,47]. The covalent tethering of specific
GPI-bound proteins to specific phospholipids may be the first event in the formation of a
lipid-domain signaling platform, or this event may occur after the domain has formed [44–47].
Depending on the cell type and cellular activity, GPI-anchored proteins in cell mem-
branes may exist in different configurations or in unique domains, and in the process, this
could result in many different, specific signaling platforms. For example, GPI-anchored
proteins in lipid domains or rafts could be involved in cell signaling, cell adhesion or
in other cellular processes [44–46,49]. Individual GPI-anchored proteins may also exist
in different modes of lateral dynamics, perhaps without any detectable movement, or
with completely free movement or free diffusion. Or these specialized domains might be
similar to some cell surface receptors and show anomalous diffusion or transiently confined
diffusion [27,31]. At the cytoplasmic membrane surface, some GPI lipid domains appear to
be dynamically capable of being linked to cortical actin-containing cytoskeletal structures.
This could explain some of the diffusion patterns seen with GPI-anchored proteins and
their spatiotemporal properties [50,51].
Cell adhesion, spreading and motility are cellular processes that may be governed
by the formation of tiny nanoclusters of small domains, some containing GPI-anchored
proteins [50,51]. For example, Mouritsen described a membrane receptor signaling pathway
that requires the formation of GPI-anchored protein nanoclusters [54]. This signaling
pathway (RhoA signaling) is initiated by the binding of extracellular proteins containing
the Arg-Gly-Asp binding motif, which can attach to cell surface β1-integrins. Binding to
β1-integrin receptors eventually activates src focal adhesion kinases at the inner membrane
surface, initiating the development of a cascade that includes actin nucleation by specific
molecules (formins) and then actin–myosin contraction, all resulting from transmembrane
linking and nanoclustering of membrane proteins. The result is a mechano-signaling
process involving direct coupling of cellular actomyosin machinery to inner cell membrane
lipids to functional GPI-anchored protein ligand-binding nanoclusters at the outer cell
membrane surface [67].
There are likely many membrane domains on outer cell membrane surfaces that can
activate specific peripheral and membrane-associated proteins at the inner cell membrane
Biomedicines 2022, 10, 1711 12 of 25

surface to form transmembrane domains, platforms or plaques in order to initiate cellular

signaling [31,54]. This process starts with ligand-binding, membrane reorganization, immo-
bilization of membrane domains, transmembrane signaling and activation of cytoplasmic
enzymes and mechanocontractile processes, and it can be used for many cell activities.
For example, it can also be used to signal internalization of plasma membrane domains
in endosomes [5,68]. These completely integrated mechano-structures exist within single
cells, groups of cells and tissues [3,5,68].
Extracellular signals from the microenvironment are constantly bombarding cells, and
these cells must have filtering mechanisms to sort out this information and pass on the
important and relevant signals to the cell’s interior. Specialized receptor structures at the cell
surface are the first level of filtering extracellular signals, followed by the need for dynamic
changes and assembly of complex signaling structures to provide additional filtering and
facilitate the transmission of signals [69]. Cell membranes, at their inner surfaces, are also
constantly interacting with and transmembrane linking to various structural components
and enzymes in order to filter, process and amplify signals from the microenvironment, and
they then pass these signals on to stimulate appropriate cellular responses. Cell membranes
are also capable of sending messages back into the extracellular environment by releasing
signaling molecules and membrane-encapsulated structures or by providing appropriate
molecular signaling patterns to adjacent cells [5].

5. The Different Distributions and Lateral Mobilities of Biomembrane Components

Cell membrane components appear to display unique rotational and lateral mobilities and
distributions due to their individual properties and a variety of restrictions on their rotational
and lateral movements. These restrictions can also affect their residence times in various com-
partments and their confinements within assorted membrane domains [3,23–25,31,37,67]. These
different lateral mobilities appear to depend on differences in local membrane compositions,
spatial organizations, linkages and obstacles that are different from the average cell-membrane
microenvironment [3,18,23,25,27,29–32,37–41,47–51,56,58–60]. For example, the lateral move-
ment of some integral cell membrane proteins in the membrane plane can be restricted by
multiple cis- and transmembrane interactions that constrain or direct their movement within or
between various membrane domains. These modulators of distribution and movement occur
within membranes, but can also include: extracellular interactions, such as binding to extracellu-
lar matrix and stroma; and intracellular interactions with peripheral and membrane-associated
cytoskeletal structures [3,18,24,25,27,31,51,54,56–60,64–67].
Biomembrane General Principle No. 14: Biomembrane components and membrane domains
display a wide spectrum of lateral movements and distributions that appear to be functionally
important and related to interactions with specific membrane domains, structures and barriers.
Biomembranes have become much more complex as data have accumulated over the
last five decades, especially on the dynamic aspects of membrane structure and function. For
example, researchers have recently discovered the presence of structural membrane barriers
that interfere with the free diffusion of membrane components in the membrane plane. At the
inner cell membrane surface, a variety of peripheral membrane barriers, such as curvature-
causing peripheral membrane proteins, cytoskeletal components and other obstacles, can
place limits on membrane component distributions and movements [3,18,20–25,27,31,56–60].
There are a variety of distinct interactions that can occur at the inner and outer cell membrane
surfaces, such as with a number of membrane complexes, domains, barriers, platforms and
membrane-associated structures [17,18,20–25,29,31,56–58,63,64].
The restraint on mobility of integral membrane glycoproteins in the cell membrane
plane and their presence in specific membrane domains has functional conseque-
nces [3,24,25,27,31,36,44,50,54,56–60,66]. The lateral movements of a few membrane pro-
teins or cell surface receptors have been examined, and their movements (or restraint of
movements) have been organized into various categories: (a) random movement or free
diffusion in the fluid portions of the membrane; (b) transient movements confined by
Biomedicines 2022, 10, 1711 13 of 25

membrane obstacles made up of protein clusters that have been likened to ‘fence posts’ or
‘pickets’; (c) transient movements that are constrained by structural domains or ‘corrals’
circumscribed by cytoskeletal elements and their attachment molecules; or (d) directed
movements due to attachment to and contraction of the cytoskeleton [24,25,27]. This has
been interpreted as various motions of membrane components as: (a) free Brownian diffu-
sion; (b) anomalous diffusion caused by changes in lipid nano-environment; (c) channeled
diffusion restrained by membrane-associated cytoskeletal structures; (d) confined diffu-
sion restrained by defined structural ‘corrals’; and (e) hop diffusion between dissimilar
domains [24,25,27,31]. For example, the original Singer–Nicolson description of integral
membrane proteins freely diffusing in the membrane plane is relevant to one of these
categories [8].
Contemporary concepts of cell membrane dynamics dictate that substantial portions
of integral membrane proteins are in mosaic structures that are incapable of free lateral
diffusion in the cell membrane plane. They may be transiently capable of undergoing
free diffusion in the membrane plane, but they are not freely mobile [18,22–25,27,28,31,63].
Some cell membrane components are thought to be wholly or partially confined to mem-
brane domains circumscribed by membrane barriers or barriers attached to the membrane
surface [22–25,27,31,40,49,55,63,65]. Since cell membranes are dynamic structures, some
integral proteins and lipids may escape from one domain and move to adjacent domains or
escape membrane domains altogether. They can also associate in the membrane plane and
become supersized mosaic structures [22,24,25,60,63]. Supersized membrane structures
may also be important in internalizing or releasing endosomes or exosomes. The abilities
of membrane lipids and proteins/glycoproteins to move between adjacent membrane
domains may be limited by the extent of their aggregation with similar or different com-
ponents, the sizes of membrane barriers to movements and the complex interactions of
membrane barriers with cytoskeletal elements and extracellular matrix or stromal compo-
nents [3,24,25,57,60].
With the realization that membrane domains are dynamic, functional structures—
the sizes of domains, their interactions, dynamics and linkages to membrane-associated
structures can vary quite dramatically, depending on a number of factors. For example,
they can exist dynamically as small lipid domains or rafts or as larger, more complex
glycoprotein–lipid–membrane-associated-cytoskeletal domains that can also be linkages
to other structures. The estimated or approximate areas of various membrane domains
can vary from 0.04 to 0.24 µm2 (these have been described as micro- and submicrometer-
sized domains). The approximate domain transit times of some membrane glycoprotein
receptors can range from 3 to 30 s. Smaller membrane domains, such as nano- or meso-
sized domains (of diameter 2–300 nm) are also present. In addition, barriers to the motion
of integral membrane proteins are present. These complex actin-containing cytoskeletal
fence domains can vary in diameter from 40–300 nm. This can be compared to small
lipid raft domains that are usually in the diameter range of 2–20 nm [24,25]. Dynamic,
integral membrane protein-complex domains are also present and can vary in size, with a
minimum range of 3–10 nm in diameter (containing only a few components) to a maximum
size of at least one hundred times this diameter [24,25]. Most cells have several different
types of cell membrane domains, and evidence suggests that some of these domains are
present as cell surface signaling complexes. This indicates that there is another, higher
level of membrane organization and complexity beyond the original description of the
Fluid-Mosaic Membrane Model [3,8,18]. Kusumi et al. [24,25] called this more-complex
representation Hierarchical Membrane Organization.
The hierarchical organization of membrane structures is based on several different ob-
servations of cell surface receptor dynamics. For example, the variability and dissimilarity
of lateral motions of various cell surface receptors and other membrane components as well
as the ability of cells to quickly change their cell surface membranes in order to respond to
intracellular and extracellular signals supports a hierarchical organization [24,25]. Thus
biomembrane organization may have evolved so that cells can rapidly and selectively
Biomedicines 2022, 10, 1711 14 of 25

respond to numerous specific extracellular signals. It may be more efficient to have various
receptors prepositioned on the cell surface within untriggered signaling domains that can
be rapidly and specifically capable of aggregating into supramolecular transmembrane
signaling structures [25]. The presence of membrane protein barriers or ‘fences’ on the
inner plasma membrane surface can limit the range of lateral motion of integral membrane
protein components. Some examples include limiting lateral motion within cytoskeletal-
fenced ‘corrals’, or tethering them directly or indirectly to membrane domains. This may
create more stable, local membrane domains of high receptor densities that do not have
to be nascently synthesized. Such hierarchical structures can incorporate membrane do-
mains with cell surface receptor diffusion rates that are 5- to 50-times lower than the same
components in freely diffusing membrane environments. Therefore, some receptors can be
confined on the average to specific membrane subregions with restricted mobilities and
ranges of display [24,25]. The prepositioning of receptors in more-dense arrays so that they
are more capable of ligand binding without requiring extensive lateral rearrangements
should increase the efficiency of response to an extracellular signal.
We can now propose that the prerequisites of some (and probably many) cell signal-
ing systems that involve cell membrane receptor–ligand binding are basic Fluid–Mosaic
membrane structures and specific membrane domains capable of forming ligand–receptor
clusters surrounded by fluid-phase lipids. In addition, many signaling domains should
be transmembrane-linked to membrane-associated signaling systems on the cytoplasmic
side of the plasma membrane [18,24,25]. A membrane signaling compartment or signaling
domain can be further defined by whether aggregations of similar or different domains
are required, or their confinement to signaling ‘zones’ by cytoskeletal or protein fencing at
the inner surface, in addition to other enzymatic properties, are important in the overall
signaling process [24,25].
Membrane receptors and their display as different arrangements in membrane do-
mains and other structures likely represents a normal situation in cell membranes in order
to accommodate the large numbers of possible extracellular signals that cells receive. In this
way, particular signals can be distinguished from one another. An example of the possible
display of hypothetical receptors in a Fluid-Mosaic membrane containing multiple lipid
domains, glycoprotein complex domains, barrier or ‘corral’ domains and other membrane-
associated structures is portrayed in Figure 2 [3,18]. Such schemes should not be taken
too seriously, because they will likely undergo further changes as soon as new data are
available. Cell membrane structures must also be dynamic and affected by a variety of
conditions, such as the binding of various extracellular molecules, changes in intra- and
extracellular ion and solute concentrations and integration of lipid molecules from inside
and outside the cell.
Cells appear to utilize different types of cell membrane domains to manage cellular
physiology. In addition to the dynamic membrane domains involved in cell signaling,
nutrient transport and other properties, cells also have less dynamic, more stable mosaic
membrane structures that are involved in preserving cell polarity, stable cell–cell inter-
actions and tissue organization. These latter properties may require more mosaic (more
structured) and less mobile receptors that are more integrated and linked to intracellular
cytoskeleton structures as well as extracellular structures in pericellular spaces. The extracel-
lular and junctional structures found between cells in tissues are also transmembrane-linked
to peripheral membrane proteins and membrane-associated cytoskeletal elements to form
integrated tissue networks. Such networks play an important role in the tensile forces and
mechanical viscoelastic responses of cells in tissues [70–72].
Biomembrane General Principle No. 15: Biomembranes undergo dynamic changes in domain
mobility, size, area and structure with assembly and disassembly of various components reacting to
changes in the microenvironment and the receiving and sending of cellular communication signals.
This general principle of biomembrane models highlights some of the organizational
and dynamic aspects of biomembranes that are difficult to present in static diagrams
Biomedicines 2022, 10, 1711 15 of 25

and figures. The greatest differences in the newer, ever-evolving Fluid–Mosaic Membrane
Model are the additions of more membrane-associated elements and the enhanced closeness
of molecular relationships or higher densities (mosaic nature) of cell membrane components.
Figure 2 depicts a rather simplified schematic of these additions to the Fluid–Mosaic Model.
As stated previously [3,18], we cannot take such schemes too seriously, because they shall
surely change again over time as more information is revealed about the structure and
dynamics of cell membranes.

6. Movements of Lipids Into/Out of Cells: Lipid Carriers, Vesicles, Globules

and Droplets
Once some of the general principles of cell membrane structure, organization and
dynamics have been established, we can ask whether these are important in the turnover
or trafficking of membrane components, such as membrane lipids. The health of tissues
and cells is highly dependent on their ability to capture and transfer nutrients and remove
cellular waste, such as toxic or damaged molecules. Once nutrients have been transported
into cells and are present in intracellular spaces, such as between various organelles and
cellular compartments, they must be delivered to various intracellular membranes and
organelles. Consequently, the abilities of cells to rapidly move various nutrients, structural
components and newly synthesized molecules to where they are needed intracellularly
and to remove them if they are damaged and no longer needed are essential to cell, tissue
and organ health. This normal trafficking of cellular molecules is especially important and
crucial for membrane lipids [73–78].
The transport of lipid molecules into cells (or their intracellular biosynthesis) and their
eventual delivery to various cell organelles and intracellular membranes (or their secretion
to the extracellular microenvironment) generally requires their movement by specific lipid
transport molecules or their incorporation into small membrane vesicles, lipid globules
or other delivery systems. These transport systems move lipids to specific intracellular
membrane sites (or the plasma membrane) or to domains at specific membrane or domain
sites [76,79,80]. Alternatively, different intracellular membranes can be used to deliver
membrane lipids to distinct membrane sites, resulting in transient fusion and exchange of
membrane constituents with other membrane compartments and organelles [79,80]. Such
processes can be used to repair damage to the plasma membrane and intracellular mem-
branes by removing damaged molecules and replacing them with undamaged molecules
in order to maintain cell function [81,82].
Biomembrane General Principle No. 16: Cells use a variety of biomembrane and related
structures, such as intracellular membranes, lipoproteins, membrane vesicles, chylomicrons and lipid
globules and droplets, to move various membrane lipids into and out of various cell compartments
and to remove damaged membrane lipids from organelles and cells.
Here we expand briefly on some of the relationships between dietary membrane lipids
and various cellular membranes, focusing on the movements of membrane lipids between
different membranes and the removal of damaged membrane lipids from cells. First, it has
been established that dietary lipid sources can be used to drive the replacement of damaged
membrane lipids, even though it is usually impossible to consume sufficient quantities
of membrane lipids in foods to fulfill this replacement by diet alone [78]. Dietary lipids,
including membrane lipids, are ingested, digested, absorbed by epithelial cells in the upper
small intestines and then transferred and transported via lymph and blood circulation to
the liver and to various organs and tissues to be absorbed again. Eventually, membrane
lipids are moved around within cells using carriers, such as lipoproteins, lipid-binding pro-
teins, chylomicrons, small lipid globules, membrane vesicles and intracellular membranes
(Figure 3) [73–76]. Inside cells, the carrier membrane vesicles, intracellular membranes,
chylomicrons and various lipid globules and droplets fuse with target membranes to de-
liver membrane lipids and to replace and remove damaged membrane lipids in a reverse
process. This entire in vivo delivery and exchange process works on a concentration gradi-
ent system by the principle of mass action or bulk flow [81]. The lipid-to-membrane and
Biomedicines 2022, 10, 1711 16 of 25

membrane-to-membrane fusion events occur continuously in cells, and these are crucial
in delivering or redistributing membrane lipids between various intracellular membranes
and between different cellular compartments.

Figure 3. A few of the phospholipid transport structures involved in the delivery of membrane
phospholipids and other lipids to intracellular membranes, and the reverse of this process to remove
damaged lipids. A liver cell is shown with internal lipid transfer and storage systems, such as lipid
micelles, globules, vesicles, chylomicrons and lipid droplets. These various lipid transport and trans-
fer structures can bind to different intracellular membranes and transfer glycerolphospholipids and
other lipids, and can pick up damaged lipids for eventual delivery to the extracellular environment.
Not shown in the figure are lipid transport/transfer by direct adjacent membrane-to-membrane
contact and lipid droplet-, globule-, chylomicron- and vesicle-to-membrane contact by temporary
fusion with adjacent intracellular membranes. Both the forward and reverse processes appear to be
driven by mass action or bulk flow mechanisms. (Modified from Nicolson et al. [83]).

The movements of lipids within cells via various lipid transport systems are quite
dependent on lipid properties. These properties include: the structure, composition, dis-
tribution and acylation of lipids in the transported lipid as well as the transport vehicle
and the target membrane domains where membrane binding and fusion occur. Other
specialized components are also required for successful lipid delivery. In addition to
the presence of fusogenic proteins, specific electrolytes and other essential molecules,
the properties of membranes at the point of fusion are also crucial [84–87]. In some
cases, lipid compositional changes can be used to track the transfer of specific lipids.
For example, the delivery of sphingolipids to specific cellular membranes can be traced
by analyzing the presence of specific sphingolipids in target membranes [48,88]. Sph-
ingolipids are commonly found to be concentrated in intracellular vesicles destined to
fuse with the plasma membrane, and thus the presence of specific membrane lipids ap-
pears to be important in the specific targeting of specialized lipids to specific membrane
domains [48,81,87].
Membrane fusion is an essential part of the process of lipid delivery to specific mem-
brane sites [84,85]. Membrane fusion is a rather common biological event, and it occurs in a
number of normal events, such as fertilization, development, endocytosis, secretion, nerve
transmission and many other normal developmental and restorative processes. Membrane
fusion is also important in many pathologic conditions, such as infection, inflammation,
neoplasia, cell death and other events [84–89].
An important restraint on membrane fusion is the necessity for specific ‘fusogenic’
proteins. Such proteins are necessary to temporarily bind adjacent membranes together
long enough for membrane fusion to take place [84,85,87,89]. This process requires the
close apposition of the two membranes, along with the presence of the fusogenic proteins
and the countering of repulsive electrostatic forces between the fusing membranes. After
close apposition, destabilization of the bilayer lipid structures occurs so that the lipids can
Biomedicines 2022, 10, 1711 17 of 25

form into a temporary non-bilayer transition structure, followed by the rapid reunification
of the membrane lipids into bilayer structures [84–87,89].
In secretory cells, the presence of dedicated fusogenic machinery at the plasma mem-
brane inner surface is notable. One example of this machinery is a specialized structure
made up of a dynamic membrane microdomain specialized for secretion, called the ‘poro-
some’ [88,90]. As visualized in electron micrographs, porosomes appear to be ‘pits’ approx-
imately 0.5–2 µm in diameter with depressions of 100–180 nm [91]. Porosomes are required
for some normal cell secretory functions, such as the secretion of proteins, glycoproteins,
enzymes, bioregulators and other important molecules [90,92].
There are other less specific membrane structures that can release lipids and other
molecules from cells. In addition to specialized plasma membrane secretory systems, cells
also spontaneously bleb and release various plasma membrane vesicles [5,93,94]. Some
of these released membrane vesicles, or exosomes, are specialized for delivery of non-
membrane molecules. Exosomes are a particular class of released membrane vesicles that
also have incapsulated soluble proteins and nucleic acids [95–97]. Thus, exosomes represent
specialized membrane vesicles with potential cell-to-cell communication properties, and
some of these may be important in the regulation of normal physiological processes [95,96].
Thus, released membrane vesicles may also be involved in the trafficking of membrane
components and the removal of damaged plasma membrane molecules from cells and
tissues [5,97].

7. Membrane Lipid Replacement with Dietary Membrane Phospholipids

As briefly discussed in the sections above, unsaturated glycerolphospholipids and other
lipids in biomembranes are eventually damaged (mainly by oxidative reactions), degraded or
destroyed and must be repaired or replaced to maintain normal cellular membrane function
and cellular physiology [3,83,98,99]. The polyunsaturated fatty acids in cellular membranes
are particularly susceptible to free radical oxidative damage, and this type of damage occurs
universally during aging and disease [98–100]. Accordingly, dietary replacement of dam-
aged membrane phospholipids with undamaged, functional phospholipids is essential in
maintaining cellular and organ function and general health [33,101–103]. However, main-
taining fully functional cellular membranes with only a dietary source for replacement of
membrane phospholipids is often quite difficult, especially in patients with chronic or acute
illnesses. Therefore, dietary supplements have been added to diets for augmenting the intake
of membrane lipids (Membrane Lipid Replacement (MLR)) [33,102,103].
Since membrane phospholipids can be oxidized, degraded or enzymatically modified
before ingestion and prior to absorption within the gastrointestinal system, the protec-
tion of membrane phospholipids before and during bioabsorption is essential for suc-
cessful MLR [104,105]. Such protection can be achieved by the addition of specific fruc-
tooligosaccharides (or inulins) to the membrane phospholipids, which directly insert into
glycerolphospholipid bilayers between adjacent head groups and protect them from ox-
idation, excess temperatures, acidity, phospholipases and bile salts [106,107]. For MLR
lipid supplements to be effective, the lipids must be protected from oxidation and degrada-
tion [33,102,103]. Because of the mass action, bulk flow principles that govern membrane
lipid transport and MLR, the purity and protection of membrane phospholipids are crucial.
When MLR phospholipids are present in excess concentrations in the gastrointestinal
system, most of these MLR phospholipids are promptly absorbed by the small intestines
as undegraded small lipid globules and micelles, not as individual molecules transported
separately. Bioabsorption of individual membrane lipid molecules is a much less efficient
process that utilizes membrane carrier or transfer proteins. Irrespective of the actual
mechanism of gut epithelial bioabsorption, the overall collective process is very efficient,
and over 90% of ingested membrane phospholipids are absorbed and transported from
the gastrointestinal system into the blood within a few hours [104,105]. While in the blood
circulation, membrane phospholipids are usually protected by their association with carrier
systems, such as lipoproteins or blood cell membranes. During MLR supplementation
Biomedicines 2022, 10, 1711 18 of 25

when membrane phospholipids are present in vast excess, they are mostly absorbed as small
phospholipid globules and micelles. Eventually the MLR phospholipids are transported
to tissues and cells, where they are transferred into cells by direct membrane contact,
endocytosis or by specific carrier and transport proteins. Once inside cells, membrane
phospholipids can be moved to various cellular compartments and organelles by a number
of mechanisms, including membrane–membrane transfer, carrier molecules, small lipid
globules, membrane vesicles, chylomicrons and other mechanisms [33,103,105]. During
this transfer process and after their intracellular delivery, the membrane lipids can be
enzymatically modified by head group exchange or by enzymatic changes in fatty acid side
chains and saturation to reflect the specific and everchanging needs of the membranes at
their final destinations [33,102,103].
As mentioned repeatedly above, the entire process of membrane lipid uptake, trans-
port, replacement, exchange and removal is driven overall by a mass action or bulk flow
mechanism [82]. Thus, when protected membrane phospholipids are in vastly excess
concentrations during MLR, they have the advantage of being able to reach their final
intracellular destinations more efficiently and with significantly less degradation or free
radical oxidation than unprotected dietary lipids. The mass action basis of bulk mem-
brane lipid uptake, transport and delivery to intracellular membranes is also true of the
reverse of this process, which eventually results in the exchange and removal of damaged,
oxidized phospholipids and their transport to and elimination via the gastrointestinal
system [74,75,77,82].

7.1. Membrane Lipid Replacement in Chronic Illnesses and Environmental Exposures

MLR, the use of oral dietary supplements containing protected, essential polyunsat-
urated glycerolphospholipids and other membrane lipids, has been successfully used to
maintain and recover lost or reduced mitochondrial and membrane function [33,102,103].
The most common dietary therapeutic use of oral MLR phospholipids is to reduce fatigue
and improve mitochondrial function [33,102,103]. Fatigue is the most common complaint of
patients seeking general medical care, and excess fatigue is associated with aging and most
if not all chronic and many acute medical conditions [108]. Fatigue is not well understood
at the cellular level; it can be perceived as a loss of overall energy, extreme mental and/or
physical tiredness, exhaustion and/or diminished endurance. It can also be apparent
as a loss of function, combined with an inability to perform even simple tasks without
exertion [108,109]. With age and in chronic and most acute diseases, fatigue is regularly
present due to a variety of causes. For example, in individuals with complaints of moderate
to severe fatigue, their fatigue has been directly related to loss of mitochondrial function
and diminished production of ATP by mitochondria [109].
Fatigue of long-term duration has been termed chronic fatigue, and this has been
seen in a variety of chronic illnesses, especially chronic fatiguing illnesses. An example
of this is chronic fatigue syndrome or myalgic encephalomyelitis (CFS/ME). CFS/ME
is a condition that features fatigue of long-term duration. Although fatigue is usually
the primary complaint, CSF/ME is a condition that involves multiple complaints and
signs and symptoms [110–112]. Moreover, in almost all chronic illnesses, fatigue is a
common secondary complaint [108,109,112]. Although severe fatigue is uniformly related
to significant loss of mitochondrial function and production of ATP, mild fatigue can be
found in depression and in some other psychiatric conditions [108,109].
MLR has been used for reducing fatigue in patients with chronic fatigue and other
chronic and fatiguing illnesses [33,102,103,112]. For example, in middle-aged to older
subjects (61–77 years-old) in a crossover clinical study, the MLR supplement NTFactor
Lipids® was used to treat chronic fatigue symptoms while mitochondrial function was
monitored. There was good correspondence between reductions in fatigue scores and
improvements in mitochondrial function tests [113]. These subjects with moderate to severe
chronic fatigue showed significant improvements in fatigue and other clinical parameters
during the test arm of the study, but the improvements were slowly reversed when patients
Biomedicines 2022, 10, 1711 19 of 25

were switched to the placebo arm of the study. The improvements in mitochondrial
function assessed by inner mitochondrial membrane transmembrane potential matched
the clinical data and showed enhancement up to 45% while on the MLR supplement, but
these gains were slowly reversed after the patients were switched to the placebo arm of
the study [113]. Similar positive results on the effects of MLR phospholipid supplements
on reducing fatigue from 26–43% were found in various chronic conditions, including
CFS/ME, fibromyalgia, Gulf War illness, chronic Lyme disease and other infections, and
various cancers [33,102,103,112–115].
Recently, we examined the ability of MLR supplements to reduce the severities of
several signs and symptoms in environmentally exposed patients [116,117]. Case reports
indicated that chemically exposed veterans with multiple-symptom conditions benefited
significantly from MLR with NTFactor Lipids® [116], so a study was initiated to examine
the effects of 6 g per day of NTFactor Lipids® on multiple signs and symptoms during
6 months of continuous MLR with oral glycerolphospholipids. The severities of over
100 signs and symptoms were reported at various times using patient illness survey forms,
and the clinical data were combined into various symptom categories [117]. This clinical
study showed that there were gradual and significant reductions of symptom severity
in categories related to fatigue, pain, musculoskeletal, nasopharyngeal, breathing, vision,
sleep, balance, urinary, gastrointestinal and chemical sensitivities after 6 months of MLR
with 6 g per day of membrane glycerolphospholipids (Figure 4). This preliminary study
indicated the potential for using MLR with membrane phospholipids to improve health
outcomes in patients with environmental exposures [117]. Although the mechanism(s) of
symptom reductions were not determined in these studies, the reductions in symptoms
might eventually be related to a combination of slow chemical removal from deeply
embedded tissue stores and enhancement of mitochondria function.

Figure 4. Symptom category scores with time reported by chemically exposed veterans who took
the oral MLR supplement NTFactor Lipids® (6 g per day) for 6 months. The mean symptom
category severity scores of all 16 trial participants (red symbols with standard error of the mean)
are compared to two individual subjects (green and blue symbols) before the trial and at one week,
one month, 3 months and 6 months. Each symptom category represents the mean of 3–6 individual
symptoms. (A) Breathing difficulties, (B) Sleep disturbances, (C) Fatigue, (D) Neurologic symptoms,
(E) Muscle symptoms, (F) Joint symptoms, (G) Vision/eye disturbances, (H) Urinary symptoms,
(I) Gastrointestinal symptoms, (J) Nasopharyngeal symptoms, (K) Pain, and (L) Chemical sensitivities.
*, p < 0.01; **, p < 0.001. Modified from Nicolson and Breeding [117].
Biomedicines 2022, 10, 1711 20 of 25

7.2. Membrane Lipid Replacement and Chronic Pain

One of the symptom categories in the studies discussed in the previous section was
pain, and MLR supplements, such as NTFactor Lipids® , have been used to help reduce
widespread musculoskeletal pain, peripheral neuropathy and gastrointestinal symptoms,
like stomach pain, in chronically ill patients [115–119]. Pain is a complex phenomenon that
can be initiated by injury, illness or environmental exposures. Pain is usually categorized
by varying criteria based on its pathophysiological mechanism, duration, etiology and
anatomical source [120,121]. One type of pain that is often widespread is nociceptive pain.
This has been described as acute or chronic pain, or as a sharp or throbbing pain that is
experienced in the joints, muscles, skin, tendons and bones. Nociceptive pain is usually
considered a short-lived condition, although it can also be chronic. This type of pain is
often generated in response to potentially harmful stimuli, and it can be divided into two
categories: somatic nociceptive pain, which is usually localized in the dermis, and visceral
nociceptive pain, which usually arises as diffuse and poorly defined pain sensations in
the midline of the body. Either type of pain can be caused by multiple events acting on
nociceptors to induce pain sensations [120,121]. The nerve membrane channels that are
involved in nociceptive pain have been identified as Transient Receptor Potential (TRP)
channels (TRPV1, TRPM3, TRPA1, etc.) [122]. This family (more than 50 subtypes) of
membrane channels has become a popular therapeutic target for the development of new
treatments for chronic pain [123].
The TRP channel superfamily in mammals consists of 6 subfamilies and 28 members
that mainly act as cation channels. These channels possess a primary structure that is
common to all of its members, and this primary structure is comprised of 6 transmembrane
domains and one hydrophilic loop that form a pore structure that is primarily permeable
to monovalent cations, but in certain cases also calcium ions [124]. Some TRP channels are
essential for nociception and thermal sensitivity [125,126].
Membrane lipids are important in the function of TRP channels. For example, some
membrane channels require the existence of phospholipids for activity [125–127]. TRP
channels are apparently regulated by membrane phosphoinositides, such as phosphatidyli-
nositol 4,5 bisphosphate or PI(4,5)P2 . Although this phospholipid was initially described as
a general inhibitor of TRP channels, it can act as an agonist with desensitization properties.
A fraction of phosphoinositol PI(4,5)P2 is present in NTFactor Lipids® , and it can inhibit
the heat- and capsaicin-activation of TRPV1 channels. The breakdown of PI(4,5)P2 by
phospholipase C alleviates this inhibition. This results in potentiation of TRPV1 activity by
proinflammatory agents such as bradykinin [128]. Even if the TRP channels are activated
by PI(4,5)P2 , they quickly become unresponsive as they become desensitized, removing the
ability to be stimulated [129].
Phospholipase C can catalyze the hydrolysis of PI(4,5)P2, resulting in the formation of
the two classical second messengers (inositol 1,4,5 trisphosphate (IP3) and diacylglycerol
(DAG)). A possible explanation for the effect of PI(4,5)P2 on TRP channels is that the
negatively charged headgroup of PI(4,5)P2 interacts with positively charged residues in
the cytoplasmic domains of TRP channels [130]. This was confirmed when the co-crystal
structures of TRP channels with and without PI(4,5)P2 were published by Hansen et al. [131].
Although the specific mechanism of action of phosphoinositosides on TRP nociceptor
channels is not fully understood, both of the proposed mechanisms (inhibition or activation
with desensitization) result in a final decrease in the activity of these channels, either
by inhibition or desensitization. The final result is a decrease in pain sensitivity and
nociception promoted by glycerolphospholipids, which are present in MLR supplements
such as NTFactor Lipids® [102,103].
The requirement of higher doses, for example 6 g per day of NTFactor Lipids® [116,117],
to significantly inhibit pain may be justified by the fact that PI and its derivatives are minor
phospholipids in MLR supplements [102,103]. Interestingly, most patients on MLR supplements,
such as oral NTFactor Lipids®, gradually moved to higher daily doses of the supplement to
control pain [116]. There are other possible explanations for the inhibition of pain by MLR
Biomedicines 2022, 10, 1711 21 of 25

supplements, such as stabilization of nerve membrane resting potential and inhibition of

depolarization. These are now under examination by the authors to determine the role of MLR
phospholipids in pain reduction.

8. Final Comment
Models of cellular membranes have evolved to be considerably more complex as well
as more compact or mosaic than the diagrams presented in the original Singer–Nicolson
Fluid–Mosaic Membrane Model [8]. Although newly published information on membrane
structure, organization and dynamics, briefly presented here in an overview, has been
generally accepted by the scientific community, we are just beginning to understand the
role of various cellular membranes and their domain properties in explaining complex
biological phenomenon. This information will be essential in elucidating the complex
interrelationships between cells in tissues and cells in fluid environments. It will also be
indispensable in the development of new therapeutic approaches, such as MLR, that can
overcome, at least in part, various pathological conditions that are linked to the loss of
cellular membrane integrity, organization, dynamics and function.

Author Contributions: Concept and original draft preparation, G.L.N.; review and editing, G.L.N.
and G.F.d.M. All authors have read and agreed to the published version of the manuscript.
Funding: G.L.N. acknowledges support from the Institute for Molecular Medicine and Nutritional
Therapeutics, Inc. G.F.d.M. has support from Research and Development Funds CSIC 91 and 137 from
the Universidad de la República, Montevideo.
Acknowledgments: We thank J. Michael for the final illustrations.
Conflicts of Interest: G.L.N. is a parttime consultant to Nutritional Therapeutics, Inc. No other
possible conflicts of interest are reported.

Abbreviations

CFS/ME chronic fatigue syndrome/myalgic encephalomyelitis

FRET Förester Resonance Energy Transfer
GPI glycosylphosphatidylinositol
MLR Membrane Lipid Replacement
PI(4,5)P2 phosphatidylinositol 4,5 bisphosphate
TRP Transient Receptor Potential

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