Download Moesin, a new cytoskeletal protein and constituent of filopodia: Its

Kidney International, Vol. 41 (1992), pp. 665—670
Moesin, a new cytoskeletal protein and constituent of
filopodia: Its role in cellular functions
HEINZ FURTHMAYR, WOLFGANG LANKES, and MANUEL AMIEVA
Department of Pathology, Stanford University Medical Center, Stanford, California, USA
Cell motility, cell attachment and cell-cell interaction are crombie et al [8, 9], to the analysis of specialized cell attachbasic cellular processes that are of fundamental importance ment sites with interference reflection microscopy [10], by
during early development, injury and repair responses or in the immunoelectron and light microscopy, of cell shape by scanprogression of tumors to metastatic disease. Cell movements ning electron microscopy [11, 12], it is clear that much has been
often occur over large distances and the cells utilize tracks learned, but these complex processes are still not understood.
(substrates) that are frequently modified, such as addition of
galactose to laminin by cell surface galactosyl transferase [1] or
proteolysis [2]. The invasion of developing tissues by other cell
types, for example, mesenchymal cells migrating into the ure-
Moesin is a member of a new family of cytoskeletal proteins
We have recently cloned and sequenced the complete cDNA
of a cellular protein with binding activity for heparin and
teral bud [3], presumably involves an invasive phase that heparan sulfate [13, 14]. We have termed this protein moesin
includes: cell-matrix and cell-cell recognition phenomena, cell (membrane organizing extension spike protein, pronounced
locomotion, and tissue degradation; a positioning phase of the [moe.ez.in]). It is a 78 kd molecule that neither contains a signal
cell, in which stable interactions are formed that presumably sequence, nor a transmembrane domain, suggesting that it is
have been intiated as a result of specific and appropriate not incorporated into the plasma membrane in the classical
cell-matrix and cell-cell contacts; and finally, a phase in which way. Its amino acid sequence is unique, but it shares sequence
the cells grow, differentiate and become polarized. Short-term identity with three other proteins: 72% with ezrin [15—18], 37%
movements often occur in development during epithelial-mes- with an N-terminal region of band 4.1 [19], and 23% with a
enchymal transformations and cell rearrangements in the early domain of talin [20]. The structural relationship with these three
embryo. These cells change their morphology from that of a cytoskeletal proteins suggests that moesin is a cytoskeletal
quiescent epithelium (polarized, stable contacts at the basal component (Fig. 1). The structure contains clustered basic
side and between cells) to a motile cell that extricates itself from residues. Such clusters have been found in other proteins that
the epithelial cell layer [4]. These cells change shape, develop bind heparin, such as some of the clotting factors [21]. They are
cell processes such as filopodia, microspikes, blebs, etc., and also seen in proteins that translocate into the nucleus (such as
eventually begin to migrate [5]. It is tempting to speculate that bFGF, [22]). This raises the question regarding the cellular
the cellular protrusions in these situations serve to probe the localization of moesin.
environment and/or the extracellular matrix (ECM), for attachCellular localization of nmesin
ment sites and other ligands [6]. In support of this idea are a
number of examples from different cell systems, in which
We prepared polyclonal antibodies to moesin and ezrin, since
receptors have been found to be concentrated in filopodia [7]. the original monoclonal antibody [14] exhibited poor tissue and
These complex developmentally regulated phenomena also cell staining. Our preliminary data indicate that cells in culture
seem to signal transitions of cells from one state to another and cannot be stained in immunofluorescence experiments with the
are transient in nature.
antibodies to moesin or ezrin, unless the cells are permeabiAnother way of looking at these phenomena is to establish lized. We have used primary cultures of endothelial (rat liver,
cells in culture and to analyze their behavior in response to human skin, mouse brain, human aorta), calf smooth muscle
obviously different conditions, such as tissue culture plastic, cells, human keratinocytes, rat astrocytes, human blood lymgrowth media, and various stimuli:chemotactic factors, growth phocytes and granulocytes, and rat liver parenchymal, Kupfer
factors, biological or other substrates, cytokines, etc. A fairly and Ito cells for these studies. We have also used cell lines
large body of information has been compiled of these attempts (HL6O, A43 1, T and B lymphocytes, intestinal and renal tubular
to not only establish often highly differentiated cells in culture, cell lines: T84, HT-29, MDCK, LLC-PK1) as well as virusbut also to understand some of the principal mechanisms transformed mouse endothelial and rat glioma cell lines (RT-2).
controlling cell behavior. Beginning with the description of In methanol or acetone fixed and permeabilized cells, a remarkvarious cellular elements involved in cell movement by Aber- able staining pattern is observed: in most of all cells grown in
tissue culture the antibodies to moesin and/or ezrin visualize
slender cell processes that vary in length depending on the cell
type and the culture conditions. In calcium depleted cultures of
© 1992 by the International Society of Nephrology
665
666
Furthmayr et a!: Moesin
mentrane
(protein)
affachrnerit
coipoin II
rttnt]èfbfl
s4e
binding
deciage
Fig. 1. Secondary structure prediction based
on the amino acid sequence of protein 4.1,
ezrin, moesin and talin. These proteins are
members of a new family and share a
homologous N-terminal domain that has been
postulated to mediate the interaction with
binding sites on the cytoplasmic face of the
plasma membrane. The central rod-like
domain has an a-helical structure and may be
involved in binding to other cytoskeletal
proteins. The C-terminal small domains are
structurally different from each other, with the
exception of moesin and ezrin, which are
closely related.
LLC-PK1 cells, for instance, they can be several cell diameters these structures of vastly differing morphology are not at all
clear and their molecular structure is largely unknown.
in length [23]. In addition to these slender structures, other
It is known from many different studies that the elaboration
types of cell processes are stained (Fig. 2). They vary somewhat
from one cell type to another. From the staining patterns it is of lamellopodia or the so-called leading edge is an extremely
clear that moesin and/or ezrin are located within these cellular rapid event that can be clearly related to cell movement [6]
projections. Frequently, moesin and ezrin are co-localized in and/or cell attachment [25]. But non-moving cells also exhibit
the same cells and the same filopodia. There are, however, a considerable surface activity. This activity refers to the protrufew examples of cells that, within our limits of detection, sion of microspikes, blebs, filopodia and related surface struccontain only one or the other protein, for example, human tures. This surface activity is not only localized to the part of
granulocytes or rat liver cells contain moesin only, while human the cell in contact with the substrate, but it is seen over the
T84 cells or chicken erythrocytes contain only ezrin.
entire surface of the cell. Significantly, it is also seen in cells
In trying to define the role of moesin, we were intrigued by that grow in suspension. Cells are thus able to produce a variety
the complexity and relative lack of clear cut definitions of the of different and rapidly "assembled" surface structures. These
various terms that have been used in the literature to describe have also been termed "motile organs" [20]. The relationship of
cell behavior and cell morphology. We have compiled names the lamellipod, a broad sheet of cytoplasm that forms the
here for surface extensions found in the literature (references leading edge of moving cells, devoid of cell organelles and
have not been included):
composed almost exclusively of actin, to the other types of cell
protrusions is also not clear. It is interesting, however, that the
filopodia, microvilli, brushborder, microspikes, prothickness of the lamellopodia is the same as the diameter of
jections, protrusions, lamellopodia, rosette, lobopodia, invadopodia, extensions, uropod, pseudopod,
trailing edge, leading edge, ruffling edge, retraction
fiber, podosome, peripheral hyaline blebs, blebs, microcolliculi, ruffle, cytekinoplast, actinoplast,
Although each of these descriptive terms refers to structures
filopodia. These other cell protrusions are frequently said to
contain actin, but the data are not precise on this point [12].
Other molecules, such as myosin, tubulin and a host of additional proteins clearly play a role in motility, but they seem to
be excluded from filopodia [26]. Changes in cell surface morphology during the cell cycle are striking in Chinese hamster
observed in specific cells and under specific conditions, they are ovary cells [11] and certain types of protrusions predominate
also used interchangeably to describe similar cellular struc- over others. Filopodial activity can be stimulated by cytokines,
tures. This has been done often in the face of clear cut evidence such as the autocrine motility factor (AMF) [27] or scatter
of discrete differences among these surface structures. One factor [28].
Lamellopodia, filopodia and other cell surface protrusions
case in point is the term microvillus, which has definitive
meaning in the context of the highly organized brush border of clearly mark active areas of the cell surface. This is in contrast
the intestinal or renal tubular epithelium with its particular to stable cell-substrate and cell-cell adhesions described as
content of fimbrin, actin, villin, etc. [24]. But the term is also close contacts, focal adhesions, ECM adhesions, or adherens
used for other cell protrusions found on many different cell junctions. While the former, in particular filopodia, microtypes that never produce a highly organized brush border, and spikes, and other membraneous structures are rapidly forming
that presumably do not contain the same set of cytoskeletal and retracting, the stable contact sites usually do not change
proteins. There are obvious differences in the appearance of unless the cell is stimulated to alter its present state, such as, to
these extensions of the cell surface in different cells as demon-
move [9]. It has been said that filopodia and other highly active
strated with antibodies to moesin. The relationship between areas of the cell surface are domains of the cell in which actin
S
667
Furthmayr et a!: Moesin
":j4
•
Fig. 2. Cellular localization of moesin. LLC-PK1 (a,b) and calf aortic smooth cells (c,d) were washed with phosphate buffer, treated with acetone
at —20°C and stained with rabbit anti-moesin and secondary FITC-labelled antibodies. In a, the cells were counter stained with propidium iodide;
in d double-staining was done with anti-vimentin and Texas red-labelled secondary antibodies. Moesin (green fluorescence) is located in cell surface
projections that vary in shape, dimension and number.
polymerization and depolymerization takes place [29]. This surface, where they occur, mark future ruffles and focal contact
might suggest that actin polymerization is responsible for the sites [25]. Since moesin is localized in all of the various surface
growth and the protrusion of the plasma membrane; electron projections, it is reasonable to postulate that it may be involved
microscopic evidence suggests, in fact, that organized microfil- in the initial protrusion, and also in the later events of convertament bundles are found in at least some, but not all filopodia ing a transient into a more permanent structure. However,
[12]. These data do not necessarily address the question moesin could also serve other functions.
Abercrombie et al. observed flow of the plasma membrane
whether actin polymerization provides the required protrusive
force rather than utilizing a previously formed structure. Bio- away from the leading edge and in a direction opposite to the
physical arguments indicate that the speed at which filopodia moving cell [8, 91. They suggested in essence that membrane is
extend and disappear is not consistent with a process of actin adsorbed in the back of the cell and then transported forward to
polymerization and depolymerization because of diffusional the leading edge of the cell, These membrane transfer cycles
limitations [30]. According to this idea, actin polymerization would enable the cell to move similar to a tank. A later
and filamentous organization does not push the filopodia out- modification of this idea suggested endocytosis as the principal
wards, but rather forms the more rigid supportive structure mechanism by which a moving cell could bring membrane
later on that allows filopodia to keep their shape. Protrusion material to the leading edge [6]. The endocytic vesicles move
could be achieved by other forces, such as an osmotic force [26, through the cytoplasm and are inserted into the plasma mem30]. This would predict that cell surface structures lacking actin brane by exocytosis at the leading front end of the motile cell.
and possibly other cytoskeletal elements exist. It has been This "tank" analogy is inadequate, however, since, while the
suggested that some of the protrusions or areas of the cell entire tank tread is carried along in its cycle, only some
668
Furthinayr et al: Moesin
Fig. 3. Tentative structural role for moesin in
microspikes. Moesin (red) in the form of
monomer or dimer interacts with binding sites
on the inner surface of the plasma membrane.
Such binding sites are provided by integrins,
CAMs and possibly other structures. This
organization may be sufficient for allowing the
formation of transient filopodia or
microspikes. "Maturation" may entail
stabilization by additional interactions with
actin and other cytoskeletal elements.
membrane components (the lipids and selected 'circulating'
proteins) participate in endocytosis. Interestingly, endocytosis
is just as active in non-motile cells as it is in motile cells. The
Function of filopodia and the relationship of moesin and/or
ezrin to these structures
major difference is that exocytosis in the non-motile cell is not
directed and that new membrane is returned randomly over the
entire surface of the cells [61. The filopodial activity we have
observed in many of our cultured cells could be a reflection of
this kind of activity. This may provide the basis for yet another
working hypothesis to explain the function of moesin: it could
We have observed that many of the cell surface protrusions
labelled with antibodies to moesin are not stained with antibodies to other cytoskeletal proteins: actin, tubulin, intermediate
filament proteins, band 4.1, fodrin (spectrin). This has also been
observed in migrating cells [1] expressing galactosyltransferase
in filopodia. First of all this suggests that these processes are
be bound to receptors, it may serve as a tag for recycling structurally heterogeneous, and secondly, that many of them
receptors, and travel with the endocytic vesicle. Filopodial may represent transient structures that are constantly forming
activity can be influenced by changes in pH, osmolarity, at a fairly rapid rate, and apparently do not require filamentous
actin for stabilization. The cell, upon stimulation, may use its
filopodial activity to assemble and concentrate receptors in
certain domains of the plasma membrane. This process may
cytokines, and other factors such as contact between cells or
certain extracellular matrix components [31]. Filopodia have
another potentially critical function, namely, to contain increased numbers of certain receptors relative to the remainder
of the cell surface.
Stable adhesion sites are domains in the plasma membrane at
which integrins and other ECM receptors interact with cytoskeletal proteins [32, 331: in focal adhesions talin provides the
link via vinculin and alpha actinin with actin filaments; in
desmosomes, intermediate filaments interact with proteins of
the plasma membrane [34]; in adherens junctions of the liver,
talin is absent, but it can be found in myotendineous junctions
and in the motor synapse. These data suggest that a variety of
structural components are required for cells to generate different stable connections. Filopodia and other cell protrusions
are usually not involved in making stable contacts. Filopodia
are said to contain receptors for extracellular ligands in significantly (10- to 20-fold) higher concentrations in comparison to
other domains of the plasma membrane [7, 27]. In certain
systems, such as in activated neutrophils, chemotactic receptors, Fe receptors, or proteases are in fact concentrated on the
leading edge of the migrating cell as is moesin. Similar observations exist in dictyostelium after a chemotactic stimulus of
membrane regions. According to this idea, moesin interaction
with the membrane and possibly with itself would precede the
polymerization of actin and other cytoskeletal assemblies (Fig.
3). It thus marks "unstable" regions of the membrane, or very
"active" regions, including those of endothelial cells that are
active in endocytosis/transcytosis.
The following points are supportive evidence for this idea.
I) Cells placed in culture respond quickly with the elabora-
cAMP has been given [35].
tion of cell protrusions [36—38]; there may be a need for de novo
allow attachment of one or more of such "arms" to the
substrate with sufficient avidity to initiate the assembly of more
permanent or more structurally stable structures. It may then
be only the latter filopodia which contain actin filaments.
Trinkaus has made the distinction between unstructured and
structured protrusions [31]. Although a number of arguments
and hypotheses have been put forward to explain protrusion of
membrane in molecular terms, there is no agreement. These
arguments have been discussed by Trinkaus [31] and Oster [30].
One of the critical questions addressed in the present article
deals with the possibility of moesin and/or ezrin to organize and
stabilize membrane receptors in such active and transient
669
Furthmayr et a!: Moesin
synthesis and/or transcription, if the particular cell type (such
as a smooth muscle cell) does not make moesin/ezrin.
2) Many of these delicate moesin/ezrin-containing cell processes cannot be stained in immunofluorescence experiments
with antibodies to a number of cytoskeletal proteins, including
fodrin, band 4.1, talin, intermediate filaments, actin, tubulin.
3) Processes are generated by the cells randomly, at least
initially, and they are not restricted to cell-matrix interaction
tures. This could be consistent with their putative role in
migration and tissue organization. In the adult rat or mouse,
moesin antibodies react predominantely with endothelial cells
in different vascular beds. This could be related to a particular
cellular activity of these cells, such as a high endocytotic or
transcytotic rate. However, co-localization in the adult animal
is seen, for instance, in the brush border of some renal tubular
segments and in glomeruli. Furthermore, there are tissues or
cell types in the adult that cannot be stained at all with either of
[36—40].
4) It has been suggested that the presence of microspikes at a the sera in tissues: smooth muscle cells in the intestine or in the
particular location of a lamellopodium or ruffling edge marks wall of blood vessels or some specialized endothelial cells.
future contacts, in which stress fibers terminate [25, 291.
Regulation of the putative interaction of moesin with the
5) Some of the cellular processes containing moesin are very
plasma membrane
long and thin, such as in calcium-depleted LLC-PK1 cells
(compare with Fig. 2). They are 20 to 100 nm in diameter and
When unstimulated A43 1 cells are treated with EGF, there is
they contact neighboring cells. "Filopodia" in these cells a rapid response of the cells within 30 seconds consisting of
apparently do not contain actin and, therefore, they may not be ruffling, the extension of filopodia and microspikes [32, 37]. The
involved in moving, pulling or attracting cells to each other. response peaks between two and five minutes, and by 10
They have been termed retraction fibers. Why they exist and minutes the ruffling subsides, and this response is followed by
why the cells apparently remain in contact with each other retraction of the cells [compare with 37]. The early response
remains unknown. In contrast, the same morphological appear- correlates with an increase in the phosphorylation of ezrin and
ance under different conditions may suggest involvement in the moesin, but there was no indication for changes in transcription
well-known phenomenon of contact inhibition of growth.
or translation. It has been shown by Bretscher and Gould et a!
6) In culture, all cells appear to express moesin and/or ezrin. [16, 17] that ezrin is phosphorylated at tyrosine and threonine
This finding is consistent with the filopodial activity observed residues. Our preliminary data indicate a similar response with
for every cell type, regardless of shape or whether the cells are respect to moesin, It is tempting to speculate that the phosphorattached to the culture dish.
ylation of these proteins is related to the early and rapid
7) Filopodial activity is initiated by cells in culture very morphological change, namely the extension of the filopodia,
rapidly (within seconds) in response to chemotactic stimuli, and that the translocation of moesin/ezrin into the newly formed
growth factors, TPA, etc. Ruffling ceases also fairly rapidly, filopodia is regulated by phosphorylation events.
when cells are placed on certain substrata.
We thus believe that moesin is involved in basic cellular
Acknowledgments
processes that are linked to cell recognition, motility, invasion,
Our research data discussed in this article were supported by grants
differentiation and cell growth. The rapidly assembled cell from the US Public Health Service (HF), a fellowship from the
surface structures may be akin to environmental sensors in a Boehringer-Inge!heim Foundation (WL) and a stipend from Stanford's
very general sense. Activity of this sort is controlled either by MSTP (MA). We also acknowledge collaborative efforts by J.F. Roll at
the environment (permissive environment activates, non-per- UCSF.
missive environment switches filopodial activity oft), or by an
intrinsic gain or loss of mobility controlled in part by expression
of the moesin and/or ezrin gene or by regulatory events. One
Reprint requests to Heinz Furthmayr, Department of Pathology,
Stanford University Medical Center, Stanford, Cahfornia 94305-5324,
USA.
would predict that mutation or loss of one or both of these genes
could result in lethal conditions, because of their importance in
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